Primary podocytes (Lonza) were maintained in RPMI medium (Capricorn) supplemented with 10% fetal calf serum (FCS). Human renal epithelial cells (HREpCs) (PromoCell) were cultured in renal epithelial cell growth medium-2 (PromoCell). Human renal glomerular endothelial cells (HRGEnC) (ScienCell) were maintained in endothelial cell medium ECM (ScienCell). Primary cells were only used from passage two to six. The human podocyte cell line was derived from human normal podocytes conditionally transformed with a temperature-sensitive mutant of the simian virus 40 (SV40) large T antigen. Cells were cultured as previously described . Experiments were performed with non-proliferating, differentiated podocytes expressing synaptopodin as podocyte-specific marker of differentiation. Vero E6 cells were maintained in DMEM (Capricorn) supplemented with 10% FCS.
Viruses and infection
Hantavirus species HTNV strain 76–118 and PUUV strain Vranica were propagated on Vero E6 cells. For infection, cells were incubated with viral inocula at a multiplicity of infection (MOI) of 0.5 and medium was replaced after six hours. MOI was calculated on the respective cell type. On day six after infection, infected cells were quantified by detection of N protein expression via immunofluorescence staining and subjected to the assays.
Human sera samples
Heat-inactivated (56 °C, 30 min) serum samples of three healthy volunteers and three patients with serologically confirmed PUUV infection were used for incubation of podocytes in migration assays. Semiquantitative proteinuria was determined by urine dipstick: +: 30 mg/dL, ++: 100 mg/dL, +++: 300 mg/dL, and ++++: 1000 mg/dL. Clinical data were collected through a review of medical charts of the Department of Nephrology of the University of Heidelberg, Germany. This study was approved by the Ethics Committee of the University of Heidelberg and it adhered to the Declaration of Helsinki. Written informed consent was obtained from all participants.
Cells grown on coverslips were fixed with 3% paraformaldehyde (PFA) and stained with primary and fluorescently-labeled secondary antibodies. The following primary antibodies were used: Mouse anti-N protein PUUV (A1C5, Progen), mouse anti-N protein HTNV (B5D9, Progen). Cell nuclei were stained by Hoechst 33342 (Invitrogen). Images were taken using an Axiocam 506 mono camera attached to an Axio Observer.D1 inverted microscope (Carl Zeiss).
For Western blot analysis, samples were boiled in SDS sample buffer, separated by SDS-PAGE, and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween20 (TBST) for 30 min at room temperature and then incubated for one hour at room temperature with the following antibodies: Mouse anti-N protein PUUV (A1C5, Progen), mouse anti-N protein HTNV (B5D9, Progen). After washing with TBST, the membrane was incubated with near-infrared fluorescently-labeled anti-mouse secondary antibodies (IRDye800, Li-Cor) for one hour at room temperature. After washing with TBST, the membrane was scanned by the Odyssey CLx infrared imaging system (Li-Cor).
Treatment of viral supernatants
Infectious hantaviral particles in supernatants derived from infected podocytes on day six after infection were inactivated by UV irradiation (1.4 J/cm2) with Stratalinker UV Crosslinker equipped with 254 nm UV-light bulbs or depleted by filtration at 5000 g at 4 °C for 2 h through Nanosep centrifugal device with Omega membrane 300 K (Pall Life Sciences) with a molecular weight cut-off of 300 kDa .
Transfection of cells
Podocytes were transfected with pmaxGFP encoding green fluorescent protein (Lonza) together with pCR3.1 (Invitrogen) encoding full-length N protein of PUUV or HTNV using nucleofection (Amaxa Nucleofector 2b device, Lonza) with the Basic Nucleofector Kit for primary mammalian epithelial cells (Lonza). Transfection of pmaxGFP together with empty vector (mock) served as control. After eight hours, cells were subjected to live cell imaging or immunofluorescence. All cells expressing maxGFP co-expressed hantaviral N protein. Percentage and viability of cells expressing max GFP together with N protein did not differ compared to cells transfected with pmaxGFP and empty vector. Amount of N protein expression was quantified by determination of the fluorescence levels of 100 transfected cells that were stained for N protein with anti-N protein antibody. Area, mean fluorescence signal, and adjacent background readings were measured for each cell using ImageJ (v1.52e, NIH). The fluorescence levels were calculated with ImageJ as total corrected cell fluorescence (TCCF) of N protein (TCCF = integrated density – (area of selected cell × mean background fluorescence)) .
Uninfected and infected cells were lysed on day six after infection. The number of viable cells was determined by measuring the amount of ATP using CellTiter-Glo luminescent cell viability assay (Promega).
Live cell imaging and single cell tracking
Infected and transfected cells (10,000 cells/cm2) on μ-slide 2-wells (Ibidi) were subjected to live cell imaging. The motility of infected and uninfected podocytes was monitored for eight hours by JuLi Smart Fluorescence Cell Imager (Digital-Bio). Cells were tracked by the ImageJ manual tracking plugin (Ibidi) and statistical analysis was done by using the chemotaxis tool plugin (Ibidi). The migration of podocytes co-transfected with N protein together with green fluorescent protein (maxGFP; Lonza) was recorded using a Ti-HCS microscope with an Andor Clara interline-CCD-camera (Nikon). Cells expressing maxGFP were tracked and analyzed as described for infected cells.
Podocytes (45,000 cells/cm2) were seeded into μ-plate wells (Ibidi) and infected with hantaviruses. The insert was removed six days after infection and images were taken immediately after insert removal and after eight hours by JuLi Smart Fluorescence Cell Imager (Digital Bio). The average area of migrated cells was measured in three independent experiments. Cells were fixed with 3% PFA and stained for nuclei and hantaviral N protein.
Uninfected or infected podocytes of a single-cell suspension (29,000 cells/cm2) were added in each well of a 96-well microtiter plate and left to adhere for one hour at 37 °C. After a triple wash with PBS, adhered cells were fixed, and stained with Sapphire700 (Li-Cor) and DRAQ5 (BioStatus) and quantified via scanning with Odyssey CLx infrared imaging system (Li-Cor).
Data were analyzed using Prism 5.0 (GraphPad Software Inc.). Normal distribution was tested with the Kolmogorov-Smirnov test. Values of two groups were compared using two-tailed Student’s t-test. Values were presented as mean ± standard deviation (SD). P values of ≤0.05 were considered significant. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns: not significant.