Kidney organoids reveal redundancy in viral entry pathways during ACE2-dependent SARS-CoV-2 infection.

With a high incidence of acute kidney injury among hospitalized COVID-19 patients, considerable attention has been focussed on whether SARS-CoV-2 specifically targets kidney cells to directly impact renal function, or whether renal damage is primarily an indirect outcome. To date, several studies have utilized kidney organoids to understand the pathogenesis of COVID-19, revealing the ability for SARS-CoV-2 to predominantly infect cells of the proximal tubule (PT), with reduced infectivity following administration of soluble ACE2. However, the immaturity of standard human kidney organoids represents a significant hurdle, leaving the preferred SARS-CoV-2 processing pathway, existence of alternate viral receptors, and the effect of common hypertensive medications on the expression of ACE2 in the context of SARS-CoV-2 exposure incompletely understood. Utilizing a novel kidney organoid model with enhanced PT maturity, genetic- and drug-mediated inhibition of viral entry and processing factors confirmed the requirement for ACE2 for SARS-CoV-2 entry but showed that the virus can utilize dual viral spike protein processing pathways downstream of ACE2 receptor binding. These include TMPRSS- and CTSL/CTSB-mediated non-endosomal and endocytic pathways, with TMPRSS10 likely playing a more significant role in the non-endosomal pathway in renal cells than TMPRSS2. Finally, treatment with the antihypertensive ACE inhibitor, lisinopril, showed negligible impact on receptor expression or susceptibility of renal cells to infection. This study represents the first in-depth characterization of viral entry in stem cell-derived human kidney organoids with enhanced PTs, providing deeper insight into the renal implications of the ongoing COVID-19 pandemic. IMPORTANCE: Utilizing a human iPSC-derived kidney organoid model with improved proximal tubule (PT) maturity, we identified the mechanism of SARS-CoV-2 entry in renal cells, confirming ACE2 as the sole receptor and revealing redundancy in downstream cell surface TMPRSS- and endocytic Cathepsin-mediated pathways. In addition, these data address the implications of SARS-CoV-2 exposure in the setting of the commonly prescribed ACE-inhibitor, lisinopril, confirming its negligible impact on infection of kidney cells. Taken together, these results provide valuable insight into the mechanism of viral infection in the human kidney.

Acute Kidney Injury in SARS-CoV-2 Infection: Direct Effect of Virus on Kidney Proximal Tubule Cells.

Coronaviruses (CoVs), including Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and the novel coronavirus disease-2 (SARS-CoV-2) are a group of enveloped RNA viruses that cause a severe respiratory infection which is associated with a high mortality [...].

Human plasmacytoid dendritic cells acquire phagocytic capacity by TLR9 ligation in the presence of soluble factors produced by renal epithelial cells.

Plasmacytoid dendritic cells (pDCs) are antigen presenting cells specialized in viral recognition through Toll-like receptor (TLR)7 and TLR9, and produce vast amounts of interferon alpha upon ligation of these TLRs. We had previously demonstrated a strong influx of pDCs in the tubulointerstitium of renal biopsies at the time of acute rejection. However, the role of human pDCs in mediating acute or chronic allograft rejection remains elusive. pDCs are thought to have a limited capacity to ingest apoptotic cells, critical for inducing CD4+ T cell activation via indirect antigen presentation and subsequent activation of antibody producing B cells. Here we tested whether the function of pDCs is affected by their presence within the graft. Maturation and interferon alpha production by pDCs was enhanced when cells were activated in the presence of viable HK2 renal epithelial cells. Importantly, soluble factors produced by cytomegalovirus-infected (primary) epithelial or endothelial cells enhanced pDC activation and induced their capacity to phagocytose apoptotic cells. Phagocytosis was not induced by free virus or soluble factors from non-infected cells. Activated pDCs showed an enhanced CD4+ and CD8+ T cell allostimulatory capacity as well as a potent indirect alloantigen presentation. Granulocyte Macrophage-Colony Stimulating Factor is one of the soluble factors produced by renal epithelial cells that, combined with TLR9 ligation, induced this functional capacity. Thus, pDCs present in the rejecting allograft can contribute to alloimmunity and potentially act as important orchestrators in the manifestation of acute and chronic rejection.

Quantitative Proteomic Analysis of Enriched Nuclear Fractions from BK Polyomavirus-Infected Primary Renal Proximal Tubule Epithelial Cells.

Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here, we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with liquid chromatography-tandem mass spectrometry. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection.

Toll like receptor 4 mediates the inhibitory effect of SARS-CoV-2 spike protein on proximal tubule albumin endocytosis.

Tubular proteinuria is a common feature in COVID-19 patients, even in the absence of established acute kidney injury. SARS-CoV-2 spike protein (S protein) was shown to inhibit megalin-mediated albumin endocytosis in proximal tubule epithelial cells (PTECs). Angiotensin-converting enzyme type 2 (ACE2) was not directly involved. Since Toll-like receptor 4 (TLR4) mediates S protein effects in various cell types, we hypothesized that TLR4 could be participating in the inhibition of PTECs albumin endocytosis elicited by S protein. Two different models of PTECs were used: porcine proximal tubule cells (LLC-PK1) and human embryonic kidney cells (HEK-293). S protein reduced Akt activity by specifically inhibiting of threonine 308 (Thr308) phosphorylation, a process mediated by phosphoinositide-dependent kinase 1 (PDK1). GSK2334470, a PDK1 inhibitor, decreased albumin endocytosis and megalin expression mimicking S protein effect. S protein did not change total TLR4 expression but decreased its surface expression. LPS-RS, a TLR4 antagonist, also counteracted the effects of the S protein on Akt phosphorylation at Thr308, albumin endocytosis, and megalin expression. Conversely, these effects of the S protein were replicated by LPS, an agonist of TLR4. Incubation of PTECs with a pseudovirus containing S protein inhibited albumin endocytosis. Null or VSV-G pseudovirus, used as control, had no effect. LPS-RS prevented the inhibitory impact of pseudovirus containing the S protein on albumin endocytosis but had no influence on virus internalization. Our findings demonstrate that the inhibitory effect of the S protein on albumin endocytosis in PTECs is mediated through TLR4, resulting from a reduction in megalin expression.

Human cytomegalovirus induces TGF-ß1 activation in renal tubular epithelial cells after epithelial-to-mesenchymal transition.

Human cytomegalovirus (HCMV) infection is associated epidemiologically with poor outcome of renal allografts due to mechanisms which remain largely undefined. Transforming growth factor-ß1 (TGF-ß1), a potent fibrogenic cytokine, is more abundant in rejecting renal allografts that are infected with either HCMV or rat CMV as compared to uninfected, rejecting grafts. TGF-ß1 induces renal fibrosis via epithelial-to-mesenchymal transition (EMT) of renal epithelial cells, a process by which epithelial cells acquire mesenchymal characteristics and a migratory phenotype, and secrete molecules associated with extracellular matrix deposition and remodeling. We report that human renal tubular epithelial cells infected in vitro with HCMV and exposed to TGF-ß1 underwent morphologic and transcriptional changes of EMT, similar to uninfected cells. HCMV infected cells after EMT also activated extracellular latent TGF-ß1 via induction of MMP-2. Renal epithelial cells transiently transfected with only the HCMV IE1 or IE2 open reading frames and stimulated to undergo EMT also induced TGF-ß1 activation associated with MMP-2 production, suggesting a role for these viral gene products in MMP-2 production. Consistent with the function of these immediate early gene products, the antiviral agents ganciclovir and foscarnet did not inhibit TGF-ß1 production after EMT by HCMV infected cells. These results indicate that HCMV infected renal tubular epithelial cells can undergo EMT after exposure to TGF-ß1, similar to uninfected renal epithelial cells, but that HCMV infection by inducing active TGF-ß1 may potentiate renal fibrosis. Our findings provide in vitro evidence for a pathogenic mechanism that could explain the clinical association between HCMV infection, TGF-ß1, and adverse renal allograft outcome.

Virological synapses allow HIV-1 uptake and gene expression in renal tubular epithelial cells.

In animal models of HIV-associated nephropathy, the expression of HIV regulatory genes in epithelial cells is sufficient to cause disease, but how the CD4-negative epithelial cells come to express HIV genes is unknown. Here, we co-cultured T cells infected with fluorescently tagged HIV with renal tubular epithelial cells and observed efficient virus transfer between these cells. The quantity of HIV transferred was much greater than that achieved by exposure to large amounts of cell-free virus and occurred without a requirement for CD4 or Env. The transfer required stable cell-cell adhesion, which could be blocked by sulfated polysaccharides or poly-anionic compounds. We found that the internalization of virus could lead to de novo synthesis of viral protein from incoming viral RNAs even in the presence of a reverse transcriptase inhibitor. These results illustrate an interaction between infected T cells and nonimmune cells, supporting the presence of virological synapses between HIV-harboring T cells and renal tubular epithelial cells, allowing viral uptake and gene expression in epithelial cells.

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Early events during BK virus entry and disassembly.

BK virus (BKV) is a nonenveloped, ubiquitous human polyomavirus that establishes a persistent infection in healthy individuals. It can be reactivated, however, in immunosuppressed patients and cause severe diseases, including polyomavirus nephropathy. The entry and disassembly mechanisms of BKV are not well defined. In this report, we characterized several early events during BKV infection in primary human renal proximal tubule epithelial (RPTE) cells, which are natural host cells for BKV. Our results demonstrate that BKV infection in RPTE cells involves an acidic environment relatively early during entry, followed by transport along the microtubule network to reach the endoplasmic reticulum (ER). A distinct disulfide bond isomerization and cleavage pattern of the major capsid protein VP1 was observed, which was also influenced by alterations in pH and disruption of trafficking to the ER. A dominant negative form of Derlin-1, an ER protein required for retro-translocation of certain misfolded proteins, inhibited BKV infection. Consistent with this, we detected an interaction between Derlin-1 and VP1. Finally, we show that proteasome function is also linked to BKV infection and capsid rearrangement. These results indicate that BKV early entry and disassembly are highly regulated processes involving multiple cellular components.

The potential effectiveness of acetazolamide in the prevention of acute kidney injury in COVID-19: A hypothesis.

Acute kidney injury (AKI) is an important complication of COVID-19 encompassing a wide range of presentations. SARS-CoV-2 is proposed to cause AKI in the patients through various mechanisms. We are, nevertheless, far from a comprehensive understanding of the underlying pathophysiological mechanisms of the kidney injury in this infection. AKI has been shown to be a marker of disease severity and also a negative prognostic factor for survival. Unfortunately, no effective preventive strategy to decrease the risk of kidney damage in these patients has yet been identified. In this hypothesis, we highlight the potential protective effects of acetazolamide, a carbonic anhydrase inhibitor, in preventing the proximal tubular damage caused by the virus through disrupting the virus-endosome fusion and also interfering with the lysosomal proteases. Our proposed mechanisms could pave the way for further in vitro studies and subsequent clinical trials.

Single-cell RNA sequencing analysis of human kidney reveals the presence of ACE2 receptor: A potential pathway of COVID-19 infection.

BACKGROUND: A novel coronavirus called SARS-Cov-2, which shared 82% similarity of genome sequence with SARS-CoV, was found in Wuhan in late December of 2019, causing an epidemic outbreak of novel coronavirus-induced pneumonia with dramatically increasing number of cases. Several organs are vulnerable to COVID-19 infection. Acute kidney injury (AKI) was reported in parts of case-studies reporting characteristics of COVID-19 patients. This study aimed at analyzing the potential route of SARS-Cov-2 entry and mechanism at cellular level. METHOD: Single-cell RNA sequencing (scRNA-seq) technology was used to obtain evidence of potential route and ACE2 expressing cell in renal system for underlying pathogenesis of kidney injury caused by COVID-19. The whole process was performed under R with Seurat packages. Canonical marker genes were used to annotate different types of cells. RESULTS: Ten different clusters were identified and ACE2 was mainly expressed in proximal tubule and glomerular parietal epithelial cells. From Gene Ontology (GO) & KEGG enrichment analysis, imbalance of ACE2 expression, renin-angiotensin system (RAS) activation, and neutrophil-related processes were the main issue of COVID-19 leading kidney injury. CONCLUSION: Our study provided the cellular evidence that SARS-Cov-2 invaded human kidney tissue via proximal convoluted tubule, proximal tubule, proximal straight tubule cells, and glomerular parietal cells by means of ACE2-related pathway and used their cellular protease TMPRSS2 for priming.

A virus-induced kidney disease model based on organ-on-a-chip: Pathogenesis exploration of virus-related renal dysfunctions.

Renal dysfunctions usually happen in viral infections and many viruses specially infect distal renal tubules, however the pathogenesis remains unknown. Here, in order to explore the pathogenesis of virus-related renal dysfunctions, a Pseudorabies Virus (PrV) induced kidney disease model was built on a distal tubule-on-a-chip (DTC), for the first time. The barrier structure and Na reabsorption of distal renal tubules were successfully reconstituted in DTCs. After PrV infection, results showed electrolyte regulation dysfunction in Na reabsorption for the disordered Na transporters, the broken reabsorption barrier, and the transformed microvilli. And it would lead to virus induced serum electrolyte abnormalities. This work brought us a new cognition about the advantages of organ-on-a-chip (OOC) in virus research, for it had given us a better insight into the pathogenesis of virus induced dysfunctions, based on its unique ability in function reproduction.

HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells.

Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis.

Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression.

The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 microg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 microg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 microg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development.

Repression of BK virus infection of human renal proximal tubular epithelial cells by pravastatin.

BACKGROUND: BK virus (BKV), a human polyomavirus, causes BKV nephritis, which often leads to graft loss after renal transplantation. Currently, the only efficient therapy against BKV nephritis seems to be a reduction or change of immunosuppressive agents, but this may increase the inherent risk of rejection. Here, we report the ability of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor (statin), which is routinely used to treat hypercholesterolemia, to repress BKV entry pathways in human renal proximal tubular epithelial cells (HRPTEC) and, correspondently, prevent BKV infection. METHODS: HRPTEC were co-incubated with BKV and pravastatin. Then the percentage of HRPTEC infected with BKV by immunofluorescent analysis and large T-antigen expression which suggested BKV infection by Western blots was assessed in the absence and presence of pravastatin. The distribution of purified and labeled BKV particles in the presence and absence of pravastatin was also investigated. RESULTS: Both the percentage of BKV infected cells and the large T-antigen expression were significantly decreased in HRPTEC pretreated and co-incubated with pravastatin. However, when pravastatin was added 72 hr after BKV infection it failed to decrease percentage of BKV infected cells. It is likely, that pravastatin's inhibitory effect is explained by depletion of caveolin-1, a critical element of caveolae. BKV enters HRPTEC by caveolar-mediated endocytosis. We provide evidence that pravastatin dramatically decreased caveolin-1 expression in HRPTEC and interfered with internalization of labeled BKV particles. CONCLUSIONS: Our data suggest that pravastatin, acting through depletion of caveolin-1, prevented caveolar-dependent BKV internalization and repressed BKV infection of HRPTEC.

Epstein-Barr virus infection of renal proximal tubule cells: possible role in chronic interstitial nephritis.

Chronic interstitial nephritis frequently accompanies renal diseases of different etiologies. Far less common is the entity of primary interstitial nephritis wherein the glomerular and vascular structures of the kidney are not the primary focus of the disease process. Using in situ hybridization and the polymerase chain reaction, we detected DNA from the Epstein-Barr Virus (EBV) exclusively in renal tissue of patients with the idiopathic variety of chronic interstitial nephritis. The EBV genome, but not that of cytomegalovirus or adenovirus, was detected primarily in renal proximal tubule cells. Furthermore, the CD21 antigen, which serves as the receptor for EBV in B lymphocytes, was detected by immunocytochemistry primarily on proximal tubule cells and was markedly upregulated in the EBV-infected tissue. Western blot analysis of primary cultures of normal proximal tubule cells identified a 140-kDa protein, confirming the expression of the CD21 antigen. Colocalization experiments using proximal and distal tubule markers confirmed that EBV DNA and the CD21 antigen are found primarily in proximal tubule cells. EBV infection of renal proximal tubular cells may participate in evoking a cellular immune response that results in a damaged renal interstitium.

Epstein-Barr Virus miR-BART6-3p Inhibits the RIG-I Pathway.

Recognition of viral pathogen-associated molecular patterns by pattern recognition receptors (PRRs) is the first step in the initiation of a host innate immune response. As a PRR, RIG-I detects either viral RNA or replication transcripts. Avoiding RIG-I recognition is a strategy employed by viruses for immune evasion. Epstein-Barr virus (EBV) infects the majority of the human population worldwide. During the latent infection period there are only a few EBV proteins expressed, whereas EBV-encoded microRNAs, such as BART microRNAs, are highly expressed. BART microRNAs regulate both EBV and the host's gene expression, modulating virus proliferation and the immune response. Here, through gene expression profiling, we found that EBV miR-BART6-3ps inhibited genes of RIG-I-like receptor signaling and the type I interferon (IFN) response. We demonstrated that miR-BART6-3p rather than other BARTs specifically suppressed RIG-I-like receptor signaling-mediated IFN-ß production. RNA-seq was used to analyze the global transcriptome change upon EBV infection and miR-BART6-3p mimics transfection, which revealed that EBV infection-triggered immune response signaling can be repressed by miR-BART6-3p overexpression. Furthermore, miR-BART6-3p inhibited the EBV-triggered IFN-ß response and facilitated EBV infection through targeting the 3'UTR of RIG-I mRNA. These findings provide new insights into the mechanism underlying the strategies employed by EBV to evade immune surveillance.

Zika virus infects renal proximal tubular epithelial cells with prolonged persistency and cytopathic effects.

Zika virus (ZIKV) infection can cause fetal developmental abnormalities and Guillain-Barré syndrome in adults. Although progress has been made in understanding the link between ZIKV infection and microcephaly, the pathology of ZIKV, particularly the viral reservoirs in human, remains poorly understood. Several studies have shown that compared to serum samples, patients' urine samples often have a longer duration of ZIKV persistency and higher viral load. This finding suggests that an independent viral reservoir may exist in the human urinary system. Despite the clinical observations, the host cells of ZIKV in the human urinary system are poorly characterized. In this study, we demonstrate that ZIKV can infect renal proximal tubular epithelial cells (RPTEpiCs) in immunodeficient mice in vivo and in both immortalized and primary human renal proximal tubular epithelial cells (hRPTEpiCs) in vitro. Importantly, ZIKV infection in mouse kidneys caused caspase-3-mediated apoptosis of renal cells. Similarly, in vitro infection of immortalized and primary hRPTEpiCs resulted in notable cytopathic effects. Consistent with the clinical observations, we found that ZIKV infection can persist with prolonged duration in hRPTEpiCs. RNA-Seq analyses of infected hRPTEpiCs revealed a large number of transcriptional changes in response to ZIKV infection, including type I interferon signaling genes and anti-viral response genes. Our results suggest that hRPTEpiCs are a potential reservoir of ZIKV in the human urinary system, providing a possible explanation for the prolonged persistency of ZIKV in patients' urine.

Caveolin- and clathrin-independent entry of BKPyV into primary human proximal tubule epithelial cells.

BK polyomavirus (BKPyV) is a human pathogen that causes polyomavirus-associated nephropathy and hemorrhagic cystitis in transplant patients. Gangliosides and caveolin proteins have previously been reported to be required for BKPyV infection in animal cell models. Recent studies from our lab and others, however, have indicated that the identity of the cells used for infection studies can greatly influence the behavior of the virus. We therefore wished to re-examine BKPyV entry in a physiologically relevant primary cell culture model, human renal proximal tubule epithelial cells. Using siRNA knockdowns, we interfered with expression of UDP-glucose ceramide glucosyltransferase (UGCG), and the endocytic vesicle coat proteins caveolin 1, caveolin 2, and clathrin heavy chain. The results demonstrate that while BKPyV does require gangliosides for efficient infection, it can enter its natural host cells via a caveolin- and clathrin-independent pathway. The results emphasize the importance of studying viruses in a relevant cell culture model.

Human endogenous retroviruses: expression in various organs in vivo and its regulation in vitro.

To evaluate the role of human endogenous retroviruses in vivo, we examined their expressions in various organs from autopsy cases by Northern blot and RT-PCR. ERV3 (HERV-R) mRNA was expressed in many organs, and the level of expression in individuals and organs considerably differed. However, expression in the adrenal gland showed consistently high levels in every individual. lambda 4-1 (HERV-E) mRNA was expressed less compared with that of ERV3, and could not be detected in the adrenal gland by Northern blot, although the expression of lambda 4-1 generally correlated with that of ERV-3 in placentas. We also examined the effect of cytokines on the transcriptional regulation of ERV3 in vitro. Although the level of ERV3 expression in cultured synovial cells did not change after IL-1 beta treatment, the level in cultured proximal tubular epithelial cells was upregulated. The evidence suggests that distinct regulatory pathways may exist for the expression of human endogenous retroviruses in different cell types.