Constitutive HO-1 and CD55 (DAF) Expression and Regulatory Interaction in Cultured Podocytes

Abstract

Overexpression of the inducible heme oxygenase (HO-1) isoform in visceral renal glomerular epithelial cells (podocytes) using in vivo transgenesis methods was shown to increase glomerular expression of the complement regulatory protein decay-accelerating factor (DAF, CD55) and reduce complement activation/deposition in a rat model of immune-mediated injury. In this preliminary study, we assessed whether constitutively expressed HO-1 regulates CD55 expression in cultured rat podocytes. We employed methods of flow cytometry, quantitative (q) RT-qPCR and post-transcriptional HO-1 gene silencing (HO-1 interfering RNA, RNAi), to assess changes in constitutive (basal) levels of podocyte HO-1 and CD55 mRNA in cultured rat podocytes. Additionally, the effect of the HO-1 inducer, heme, on HO-1 and CD55 expression was assessed. Results indicate that rat podocytes constitutively express HO-1 and DAF and that the HO-1 inducer, heme, increases both HO-1 and DAF expression. HO-1 gene silencing using RNA interference (RNAi) is feasible but the effect on constitutive CD55 transcription is inconsistent. These observations are relevant to conditions of podocyte exposure to heme that can activate the complementary cascade, as may occur in systemic or intraglomerular hemolysis.

Keywords: CD55; DAF; HO-1; hemin; podocytes; siRNA.

Figure 1

Cytotoxicity of heme (hemin formulation) assessed by the lactate dehydrogenase (LDH) release assay. Media samples from each flask were removed and diluted into LDH assay buffer. LDH activity was measured by combining 50 μL diluted sample with 50 μL LDH Detection Reagent. Relative Luminescence (RLU) of each sample was measured using a GloMax Luminometer (Promega, Madison, WI, USA) after a Linear Range of LDH positive control standard curve was constructed to assess linearity of the assay at various concentrations (left panel). Raw data (LDH release), measured in luminescence units, following 18 h incubations with hemin (0, 5, 10, 50 and 100 µM) are shown in right panel.

Figure 2

Cytotoxicity of heme (hemin) assessed by live cell morphology. Cells were plated in DMEM media with 10% FBS and incubated with varying concentrations (0, 5, 10, and 50 µM) of hemin for 18 h. Changes in morphology was assessed using an ImageXpress Pico Microscopy system. No change in podocyte morphology was observed at concentrations below 50 µM (panels a–c). Concentrations on 0, 5 and 10 µM were, therefore, deemed as non-cytotoxic. Cell rounding was observed at hemin concentration of 50 µM (d).

Figure 3

(a) Flow cytometry image gallery: Constitutive HO-1 and CD55 expression in podocytes. Cultured podocytes were fixed and permeabilized with 4% formaldehyde solution. Cells were then directly stained with an AF (Alexa Fluor) 647-conjugated anti-rat HO-1 antibody at a 1250-fold dilution and a FITC-conjugated anti-rat CD55 antibody at a 2000-fold dilution. Antibody incubations were for 30 min at 22 °C followed by flow cytometry using the Amnis FlowSight imaging cytometer (Luminex Corporation, Austin, TX, USA) that detects brightfield cell morphology (channels 01 and 09 in figure above), darkfield and fluorescent images (FITC in channel 02, AF647 in channel 11 in figure above). Side scatter, a measure of internal complexity (i.e., granularity) of interrogated cells, was also assessed (channel 06). Events captured were 5000 for samples with either AF647-conjugated anti-rat HO-1 antibody or FITC-conjugated anti rat CD55 antibody, 1000 in compensation sample for each of these conjugated antibodies and 5000 for unstained sample. Constitutive expression of both HO-1 and CD55 was observed (channels 02 and 11). (b) Histogram showing results of flow cytometry analysis: intensity of staining of podocytes incubated with FITC-conjugated anti-rat CD55 antibody (left panel) and of AF647 conjugated anti-rat HO-1 antibody (right panel) plotted against number of cells interrogated.

Figure 4

(a) Flow cytometry image gallery: Assessment of HO-1 inducibility by heme in cultured podocytes. At completion of incubations with heme (0, 5 and 10 µM for 24 h), cells were fixed and permeabilized with 4% formaldehyde solution followed by direct staining (30 min at 22 °C) with AF647 conjugated anti-rat HO-1 antibody at 1250-fold dilution and flow cytometry. Channels 01 and 09 in figure above: bright field cell imaging/morphology; Chanel 11: cells stained with the AF647 conjugated anti-HO-1 antibody; Channel 06: side scatter (SSC) reflecting internal complexity/morphology of interrogated cells. A total of 5000 events were captured for each of AF647-conjugated anti-rat HO-1 antibody-stained samples and for unstained sample (control). Heme concentration-dependent increase in HO-1 expression was observed. (b) Composite histogram representing gated cell populations. Gray histogram: podocytes not stained with the anti-rat HO-1 antibody. Turquoise histogram: podocytes incubated with 0 µM of hemin and stained with the anti-rat HO-1 antibody. Purple histogram: podocytes incubated with 5 µM of hemin and stained with the anti-rat HO-1 antibody. Green histogram: podocytes incubated with 10 µM of hemin and stained with the anti-rat HO-1 antibody. Shift of histogram curve to the right in response to increasing hemin concentrations indicative of HO-1 induction.

Figure 5

Effect of sub-cytotoxic heme concentrations on HO-1 and CD55 DAF gene expression relative to that of GAPDH expressed as fold change using the ΔΔCq (Livak) method. Total RNA (5 µg) from podocytes incubated with heme (0, 5, 10 µM for 24 h) was reverse transcribed to cDNA to perform RT-qPCR on 10-fold dilution duplicates of the cDNA template. Bar graphs show a dose-dependent effect of hemin resulting in increased gene expression of both CD55 (DAF) and HO-1.

Figure 6

(a) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte HO-1 mRNA transcripts. Transfections were performed with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes as described in Methods. Results were expressed as percent remaining HO-1 mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex (results of one transfection experiment for each HO-1 siRNA duplex are shown). Negative control (non-targeting siRNA, a commercially available siRNA Silencer Select Negative Control No. 1 siRNA used to control for non-specific effects related to siRNA delivery) was taken as 100% gene expression. Transfection with HO-1 siRNA duplexes resulted in a concentration-dependent decrease in HO-1 mRNA transcripts which were undetectable at duplexes concentration of 50 nM. (b) Effect of HO-1 RNA interference (HO-1 RNAi) on constitutive level of podocyte CD55 (DAF) mRNA transcripts. Results were expressed as percent remaining DAF mRNA transcripts for each of 10, 30 and 50 nM concentrations of HO-1 siRNA duplex. HO-1 siRNA duplexes at 10 nM concentration had no effect. At 30 and 50 nM concentrations, constitutive level of CD55 (DAF) transcripts reduced, but this effect was inconsistent.

Figure 7

Effect of constitutive HO-1 silencing of basal CD55 (DAF) mRNA levels. In podocytes transfected with HO-1 RNAi duplex-Lipofectamine RNAiMAX complexes to silence HO-1, there was no statistically significant change in CD55 (DAF) mRNA.

Figure 8

Detection of changes in constitutive DAF (CD55) and HO-1 protein levels, assessed by Western blot analysis of total protein extracts obtained from two separate podocyte cultures (panel A), and in CD55 mRNA levels assessed by RT-PCR (panel B) in podocytes transfected with HO-1 siRNA. In the duplicate experiment (panel A), bands in lanes 1 (first experiment) and 4 (second experiment) reflect level of constitutively expressed HO-1 protein in untransfected (control, Ctr) podocytes. Transfection with increasing concentrations of HO-1 RNAi duplexes (siHO-1 RNA) reduced HO-1 protein levels in a duplex concentration-dependent manner (lanes 2 and 3 in first experiment; lanes 5 and 6 in the second experiment) compared to levels in untransfected cells (Ctr, lanes 1 and 4). There was no effect of HO-1 silencing on CD55 (DAF) mRNA (B).

Figure 9

Working hypothesis cartoon. In podocytes, there is a regulatory interaction between HO-1 and CD55 whereby HO-1 preserves or increases CD55. While this effect was shown in whole glomeruli with podocyte-targeted HO-1 overexpression [4], it remains to be shown in isolated (cultured) podocytes. In these, constitutive HO-1 gene expression can be efficiently silenced without a significant effect on basal CD55 expression in the absence of heme exposure. The effect of HO-1 silencing on CD55 expression in the presence of heme remains to be shown.