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

ABSTRACT: 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.

SUBMITTER: Chen J

PROVIDER: S-EPMC5583673 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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

Chen Jian J Yang Yi-Feng YF Chen Jun J Zhou Xiaohui X Dong Zhaoguang Z Chen Tianyue T Yang Yu Y Zou Peng P Jiang Biao B Hu Yunwen Y Lu Lu L Zhang Xiaoyan X Liu Jia J Xu Jianqing J Zhu Tongyu T

Emerging microbes & infections 20170823 8

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 ...[more]

PMID: 28831192

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Project description:BK virus (BKV; human polyomavirus 1) infections are asymptomatic in most individuals, and the virus persists throughout life without harm. However, BKV is a threat to transplant patients and those with immunosuppressive disorders. Under these circumstances, the virus can replicate robustly in proximal tubule epithelial cells (PT). Cultured renal proximal tubule epithelial cells (RPTE) are permissive to BKV and have been used extensively to characterize different aspects of BKV infection. Recently, lines of hTERT-immortalized RPTE have become available, and preliminary studies indicate they support BKV infection as well. Our results indicate that BKV infection leads to a similar response in primary and immortalized RPTE. In addition, we examined the patterns of global gene expression of primary and immortalized RPTE and compared them with uncultured PT freshly dissociated from human kidney. As expected, PT isolated from the healthy kidney express a number of differentiation-specific genes that are associated with kidney function. However, the expression of most of these genes is absent or repressed in cultured RPTE. Rather, cultured RPTE exhibit a gene expression profile indicative of a stressed or injured kidney. Inoculation of cultured RPTE with BKV results in the suppression of many genes associated with kidney stress. In summary, this study demonstrated similar global gene expression patterns and responses to BKV infection between primary and immortalized RPTE. Moreover, results from bulk transcriptome sequencing (RNA-seq) and SCT experiments revealed distinct transcriptomic signatures representing cell injury and stress in primary RPTE in contrast to the uncultured, freshly dissociated PT from human kidney. IMPORTANCE Cultured primary human cells provide powerful tools for the study of viral infectious cycles and host virus interactions. In the case of BKV-associated nephropathy, viral replication occurs primarily in the proximal tubule epithelia in the kidney. Consequently, cultured primary and immortalized renal proximal tubule epithelial cells (RPTE) are widely used to study BKV infection. In this work, using bulk and single-cell transcriptomics, we found that primary and immortalized RPTE responded similarly to BKV infection. However, both uninfected primary and immortalized RPTE have gene expression profiles that are markedly different from healthy proximal tubule epithelia isolated directly from human kidney without culture. Cultured RPTE are in a gene expression state indicative of an injured or stressed kidney. These results raise the possibility that BKV replicates preferentially in injured or stressed kidney epithelial cells during nephropathy.

Project description:We examined the roles of Notch1 signaling and its cross-talk with other signaling pathways, including p53 and phosphatidylinositol-3-kinase (PI3K)/Akt, in cadmium-induced cellular damage in HK-2 human renal proximal tubular epithelial cells. Following exposure to cadmium chloride (CdCl2), the level of Notch intracellular domain (NICD), the cleaved form of the Notch1 receptor, was increased and accumulated in the nuclear fraction. Knockdown of Notch1 with siRNA or treatment with the ?-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester), prevented CdCl2-induced morphological change of HK-2 cells and reduction of cell viability. Knockdown of Jagged1 or Jagged2, the ligands of the Notch1 receptor, partially suppressed cadmium cytotoxicity. Inhibition of p53 activity with pifithrin-? or inhibition of PI3K with LY294002 suppressed CdCl2-induced cellular damage and elevation of Notch1-NICD. In addition, treatment with the epidermal growth factor receptor (EGFR) inhibitor, AG1478, and the insulin-like growth factor-1 receptor inhibitor, PPP, suppressed both Notch1-NICD accumulation and Akt phosphorylation in HK-2 cells exposed to CdCl2. However, knockdown of Notch1 did not affect CdCl2-induced p53 accumulation and phosphorylation but suppressed phosphorylation of EGFR, Akt, and p70 S6 kinase. Depletion of Notch1 suppressed CdCl2-induced reduction of E-cadherin expression and elevation of Snail expression. Furthermore, treatment with SB216763, an inhibitor of glycogen synthase kinase-3, suppressed the potency of LY294002 treatment to reduce Snail expression in HK-2 cells exposed to CdCl2. Knockdown of Snail with siRNA partially prevented HK-2 cells from CdCl2-induced reduction of E-cadherin expression and cellular damage. These results suggest that cadmium exposure induces the activation of Notch1 signaling in renal proximal tubular cells with cooperative activation by the p53 and PI3K/Akt signaling pathways; the resultant expression of Snail, a repressor of E-cadherin expression, might lead to cellular damage by decreasing cell-cell adhesion.

Project description:BackgroundEverolimus (EVE) is a drug widely used in several renal transplant protocols. Although characterized by a relatively low nephrotoxicity, it may induce several adverse effects including severe fibro-interstitial pneumonitis. The exact molecular/biological mechanism associated to these pro-fibrotic effects is unknown, but epithelial to mesenchymal transition (EMT) may have a central role. Additionally, heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells.MethodsSeveral biomolecular strategies (RT-PCR, immunofluorescence, zymography and migration assay) have been used to assess the capability of EVE (10, 100, 200 and 500 nM) to induce an in vitro heparanase-mediated EMT in wild-type (WT) and Heparanase (HPSE)-silenced immortalized human renal epithelial proximal tubular cells (HK-2). Additionally, microarray technology was used to find additional biological elements involved in EVE-induced EMT.ResultsBiomolecular experiments demonstrated a significant up-regulation (more than 1.5 fold increase) of several genes encoding for well known EMT markers [(alpha-smooth muscle actin (?-SMA), Vimentin (VIM), Fibronectin (FN) and matrix metalloproteinase-9 (MMP9)], enhancement of MMP9 protein level and increment of cells motility in WT HK2 cells treated with high concentrations of EVE (higher than 100 nM). Similarly, immunofluorescence analysis showed that 100 nM of EVE increased ?-SMA, VIM and FN protein expression in WT HK2 cells. All these effects were absent in both HPSE- and AKT-silenced cell lines. AKT is a protein having a central role in EMT. Additionally, microarray analysis identified other 2 genes significantly up-regulated in 100 nM EVE-treated cells (p?<?0.005 and FDR?<?5%): transforming growth factor beta-2 (TGF?2) and epidermal growth factor receptor (EGFR). Real-time PCR analysis validated microarray.ConclusionsOur in vitro study reveals new biological/cellular aspects of the pro-fibrotic activity of EVE and it demonstrates, for the first time, that an heparanase-mediated EMT of renal tubular cells may be activated by high doses of this drug. Additionally, our results suggest that clinicians should administer the adequate dosage of EVE in order to increase efficacy and reduce adverse effects. Finally heparanase could be a new potential therapeutic target useful to prevent/minimize drug-related systemic fibrotic adverse effects.

Project description:4-Hydroxy-2-hexenal (HHE), the aldehyde product of lipid peroxidation, may be responsible for the pathogenesis of progressive renal disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to be renoprotective through its anti-inflammatory and antifibrotic effects in various experimental nephropathy models. In this study, we investigated the effects of paricalcitol on inflammation and epithelial-mesenchymal transition (EMT) after HHE-induced renal tubular epithelial cell injury. To investigate the molecular mechanisms underlying HHE-induced renal tubular cell injury, the human proximal tubular epithelial (HK-2) cells cultured with 10 µM HHE in the presence or absence of paricalcitol. In HK-2 cells, paricalcitol attenuated the HHE-induced expression of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, and prevented nuclear factor-κB (NF-κB) activation. The expression of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 was attenuated by paricalcitol pretreatment. In addition, HHE increased the expression of the transforming growth factor (TGF)-β/Smad signaling proteins and fibrotic proteins, such as α-smooth muscle actin and connective tissue growth factor; this inducible expression was suppressed by pretreatment with paricalcitol. Treatment with HHE resulted in the activation of the β-catenin signaling pathway, and paricalcitol pretreatment reduced the expression of β-catenin in HHE-treated HK-2 cells. Coimmunoprecipitation shows that paricalcitol induced vitamin D receptor (VDR)/β-catenin complex formation in HK-2 cells. Also immunofluorescence staining revealed that co-localization of VDR and β-catenin in the nuclei. ICG-001, an inhibitor of β-catenin, decreased the expression of TGF-β1 and attenuated HHE-induced tubular EMT. These results show that paricalcitol attenuated HHE-induced renal tubular cell injury by suppressing inflammation and EMT process through inhibition of the NF-κB, TGF-β/Smad, and β-catenin signaling pathways.

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

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

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