Hepatitis E Virus Mediates Renal Injury via the Interaction between the Immune Cells and Renal Epithelium

Abstract

Renal disorders are associated with Hepatitis E virus (HEV) infection. Progression to end-stage renal disease and acute kidney injury are complications associated with HEV infection. The mechanisms by which HEV mediates the glomerular diseases remain unclear. CD10⁺/CD13⁺ primary proximal tubular (PT) epithelial cells, isolated from healthy donors, were infected with HEV. Inflammatory markers and kidney injury markers were assessed in the presence or absence of peripheral blood mononuclear cells (PBMCs) isolated from the same donors. HEV replicated efficiently in the PT cells as shown by the increase in HEV load over time and the expression of capsid Ag. In the absence of PBMCs, HEV was not nephrotoxic, with no direct effect on the transcription of chemokines (Cxcl-9, Cxcl-10, and Cxcl-11) nor the kidney injury markers (kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and interleukin 18 (lL-18)). While higher inflammatory responses, upregulation of chemokines and kidney injury markers expression, and signs of nephrotoxicity were recorded in HEV-infected PT cells cocultured with PBMCs. Interestingly, a significantly higher level of IFN-γ was released in the PBMCs-PT coculture compared to PT alone during HEV infection. In conclusion: The crosstalk between immune cells and renal epithelium and the signal axes IFN-γ/chemokines and IL-18 could be the immune-mediated mechanisms of HEV-induced renal disorder.

Keywords: HEV; IFN-γ; chemokines; immune-mediated; inflammatory cytokines; kidney injury; proximal tubular; renal disorder.

Figure 1

Isolation and characterization of primary human proximal tubular (PT) epithelial cells. (A) Schematic flow showing the isolation of PT epithelial cells from a kidney biopsy, and sorting of the cells using microbeads to isolate CD10⁺/CD13⁺ cells. (B) The isolated human PT cells were analyzed by flow cytometry to check for purity and the isolated cells were stained with anti-CD10 and anti-CD13. (C) The isolated human PT cells were checked for MUC-1, E-cadherin, Aquaporin1, and ICAM-1 mRNA expression by qPCR. Black columns represent unsorted cells, and grey columns represent CD10⁺/CD13⁺ cells. Depicted are the mean values of three independent experiments ± SEM. *** indicates p < 0.001 as determined by student’s t-test. (D) Transepithelial electrical resistance (TEER) was measured in the cultured CD10⁺/CD13⁺ PT cells over time.

Figure 2

Infection of the CD10⁺/CD13⁺ PT epithelial cells with HEV inoculum. (A) Infection of polarized CD10⁺/CD13⁺ PT cells, cultured on transwell, with HEV-1 inoculum at the basolateral side. Intracellular (dotted line) and extracellular (solid line) HEV RNA was quantified by qPCR. LOQ: limit of quantification. Depicted are the mean values of three independent experiments ± SEM. (B) Representative gating strategy showing the expression of HEV ORF2 Ag in the PT cells infected with HEV. The dark histogram represents cells stained with the secondary A488 conjugated anti-mouse antibodies alone; the light shaded histogram represents cells stained by mouse anti-HEV-ORF2 followed by A488 conjugated anti-mouse antibody. (C) Supernatants collected from HEV-1 infected PT cells were tested for HEV ORF2 Ag by ELISA, C.O is the cutoff. Depicted are the mean values of three independent experiments ± SEM. (D) HEV RNA (black), and HEV Ag (blue) were measured at the basolateral and apical sides. Depicted are the mean values of three independent experiments ± SEM.

Figure 3

HEV infection alone did not affect the transcription of chemokines nor kidney injury markers of the PT epithelium. CD10+/CD13+ PT cells were challenged with HEV-1 for 10 days and then total cellular RNA was extracted and the mRNA expression level of proinflammatory markers (IL-8, IL-6, MCP-1, TNF-α, and IL-1β) (A), chemokines (Cxcl-9, Cxcl-10, and Cxcl-11) (B), and kidney injury transcripts (kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and interleukin 18 (lL-18)) (C) were assessed. The relative gene expression was determined by comparing the expression levels of these transcripts with mock cells. Black columns represent uninfected cells, and grey columns represent HEV infected cells. Data represent the mean +/− SEM of four separate experiments. *, **, indicates p ≤ 0.05 and p ≤ 0.01 as assayed by two-tailed Student’s t-test. (D) The level of LDH was measured in the supernatants of PT infected or not with HEV at Day 10 postinfection. Data represent the mean ± SEM of four separate experiments. Black columns represent uninfected cells, and grey columns represent HEV infected cells.

Figure 4

Coculture of PBMCs with the PT epithelial cells increased the inflammatory and kidney injury markers produced from the epithelium upon HEV infection. CD10⁺/CD13⁺ PT cells were infected with HEV-1 for 7 days and then PBMCs from the same donors were added for an additional 3 days. Total cellular RNA was extracted from the PT cells and the mRNA expression level of proinflammatory markers (IL-8, IL-6, MCP-1, TNF-α, and IL-1β) (A), chemokines (Cxcl-9, Cxcl-10, and Cxcl-11) (B), and kidney injury transcripts (KIM-1, NGAL, and IL-18) (C) were assessed. The relative gene expression was determined by comparing the expression levels of these transcripts with mock cells (PT + PBMCs). Black columns represent uninfected cocultured PT/PBMCs cells, and grey columns represent HEV infected PT/PBMCs cocultured cells. Data represent the mean ± SEM of four separate experiments. *, **, indicates p ≤ 0.05 and p ≤ 0.01 as assayed by two-tailed Student’s t-test. (D) LDH assay was performed on PT cells either uninfected or HEV-infected in the presence or absence of coculture with PBMCs.

Figure 5

Exacerbation of the inflammatory response was dependable on IFN-γ produced from the PBMCs. PT cells, either uninfected or HEV-infected in the presence or absence of coculture with PBMCs, were assessed for IFN-γ. The level of IFN-γ was measured in the apical (upper compartment) (A) and basolateral (lower compartment) (B) of PT cells, PT cells cocultured with PBMCs challenged or not with HEV. PT cells were treated or not with IFN-γ (1 ng/mL) for 3 days. The relative expression levels of kidney injury markers (KIM-1, NGAL, and IL-18) (C) and chemokines (D) was compared in PT cells treated (gray) or not (black) with IFN-γ. Data represent the mean ± SEM of four separate experiments. *, **, and *** indicates p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001 as assayed by two-tailed Student’s t-test.

Figure 6

The crosstalk between the renal epithelium and PBMCs following HEV infection. Schematic showing the crosstalk between the epithelium and the immune cells (PBMCs) and the interplay between IFN-γ, chemokines, and IL-18 are the main mediators of HEV-induced renal epithelium damage.