Sublytic C5b-9 Complexes Induce Apoptosis of Glomerular Mesangial Cells in Rats with Thy-1 Nephritis through Role of Interferon Regulatory Factor-1-dependent Caspase 8 Activation^*

Reagents and Animals

A monoclonal antibody against IRF-1 was purchased from BD Biosciences. Polyclonal antibodies against procaspase/cleaved caspase 9, procaspase/cleaved caspase 8, procaspase/cleaved caspase 3, and a Western blot detection system were purchased from Cell Signaling Technology (Beverly, MA). The chromatin immunoprecipitation (ChIP) assay kit was from Millipore (Bedford, MA). The specific inhibitors of caspase 8 (Z-IETD-FMK) and caspase 3 (Z-DEVD-FMK) were purchased from R&D Systems (Minneapolis, MN). pRL-SV40 luciferase reporter was provided by Promega (Madison, WI). pGL3-basic vector and a 1.7-kb rat IRF-1 promoter were provided by Dr. Fangming Tang (University of Chicago) and Dr. Li-yuan Yu-Lee (Baylor College of Medicine, Houston, TX). SD rats were from B&K Universal Ltd. (Shanghai, China). Institutional approval for the animal study protocol was obtained. The rat GMC strain was provided by the China Center for Type Culture Collection (Wuhan, China). Normal serum (NS) from 10 healthy adult donors, and heat-inactivated serum (HIS) was obtained by incubating NS at 56 °C for 30 min. Human complement C6-deficient serum (C6DS) was obtained from Sigma. Rabbit polyclonal antibody against Thy-1 antigen of rat thymocyte (anti-Thy-1 Ab) was prepared according to previously published procedures (6, 7).

IRF-1 Plasmid Construction

The expression plasmid of pcDNA3.1/IRF-1 (pIRF-1) was constructed by inserting the complete ORF of rat IRF-1 cDNA into the expression vector (pcDNA3.1). In brief, the first strand cDNA was synthesized from total RNA of cultured GMC using M-MLV-RT (14). The IRF-1 gene was amplified by PCR. Specific primer sequences were as follows: forward primer, 5′-GCGCGGATCCATGCCTATCACTCGGATGCGAATG-3′; reverse primer, 5′-GCGCAAGCTTTGGTGCACAAGGAATAGCCTGA-3′.

IRF-1 shRNA Vector Generation

Three classes of different shRNA sequences were designed to be homologous to the Rattus norvegicus IRF-1, complete CDS (GenBank^TM accession number NM_012591.1). The complementary oligonucleotides encoded a hairpin structure with a 21-mer stem derived from the target site. The vectors of shIRF-1-expressing plasmid were constructed using pGCsi as the vector backbone, whereas a scrambled gene shRNA (control shRNA) was used as a negative control (14, 15).

Caspase 8 Promoter Luciferase Reporter Construction

The luciferase reporter was constructed by inserting the 1.24-kb caspase 8 promoter (nt −1136 to +101) into pGL3-basic vector. This construct contains nucleotides 57, 417, 453–57, 418, and 689 from the reference genomic sequence NC_005108.2 and corresponds to nucleotides −1136/+101 of the rat caspase 8 gene. The 1237-bp promoter of the rat caspase 8 gene was amplified by PCR: forward primer, 5′-GCGCGGTACCCCTTGGCTTGTTGTCA-3′; reverse primer, 5′-GCGCAAGCTTTTCTTCTGCGGGATGT-3′ (the restriction enzyme cutting site is underlined). To determine the minimal caspase 8 promoter sequence required for constitutive and inducible activity, we constructed the following promoter deletion fragments by PCR and cloned them into the same reporter vector: nt −936 to +101, −736 to +101, −336 to +101, and −136 to +101. Specific primers for different promoter deletion fragments were as follows: 1) nt −936 to +101 of rat caspase 8 gene (forward primer, 5′-GCGCGGTACCAGTGGACAGGTCTGTG-3′; reverse primer, 5′-GCGCAAGCTTTTCTTCTGCGGGATGT-3′); 2) nt −736 to +101 of rat caspase 8 gene (forward primer, 5′-GCGCGGTACCGACTCTATAAGCTATA-3′; reverse primer, 5′-GCGCAAGCTTTTCTTCTGCGGGATGT-3′); 3) nt −336 to +101 of caspase 8 gene (forward primer, 5′-GCGCGGTACCCACTTTTCCTTTAAGA-3′; reverse primer, 5′-GCGCAAGCTTTTCTTCTGCGGGATGT-3′); 4) nt −136 to +101 of the rat caspase 8 gene (forward primer, 5′-GCGCGGTACCTCTAAAGTGTCTGGAA-3′; reverse primer, 5′-GCGCAAGCTTTTCTTCTGCGGGATGT-3′).

GMC Culture and Sublytic C5b-9 Determination

To ensure that C5b-9 attack was insufficient to lead to cell lysis, lactate dehydrogenase was detected in the supernatants of cultured GMC using a lactate dehydrogenase assay kit, and less than 5% lactate dehydrogenase release from cells was regarded as a sublytic effect (14, 15). Based on a checkerboard titration test, 5% Thy-1 Ab and 4% NS were used to form sublytic C5b-9 complexes (14). To ascertain that the effects on GMC were due to formation and attack of sublytic C5b-9, GMC were also treated with anti-Thy-1 Ab + C6DS, anti-Thy-1 Ab + HIS, anti-Thy-1 Ab, and MEM as control groups (14, 15).

GMC Transfection and Identification

Transient transfection of pcDNA3.1/IRF-1 (pIRF-1) or IRF-1 shRNA expression plasmid into cultured GMC was conducted with GenEscort^TM III. The IRF-1 protein expression was assessed by Western blot (supplemental Fig. 1A), and the efficiency of shRNA transfection was examined by green fluorescence protein (GFP) expression in supplemental Fig. 1B. As for IRF-1 shRNA, the three classes of shRNA to IRF-1, different target sites were designed and transfected into GMC to repress the target gene. The shRNA-2 (caGCTCTACTCTGCCTGATGATTCAAGAGATCATCAGGCAGAGTAGAGCtg) that could effectively silence IRF-1 expression (supplemental Fig. 1C) was chosen.

Thy-1N Model Reproduction and Experimental Design

Normal male SD rats (160–200 g) were randomly divided into two groups (n = 8): 1) Thy-1N group (rats were given anti-Thy-1 Ab (0.75 ml/100 g body weight) by a single intravenous injection; 2) NS group (rats were injected intravenously with normal rabbit serum (0.75 ml/100 g body weight). Samples of renal cortexes were obtained at 0 min, 20 min, 40 min, 80 min, 3 h, 6 h, and 12 h after the administration of serum by biopsies or sacrifice (6). Some samples obtained at fixed times were first examined using microarray, real-time PCR, and Western blot for the expressions of IRF-1 and caspases 9, 8, and 3.

In order to find the most optimal time of IRF-1 shRNA transfer, the plasmids were transferred into rat kidneys via renal artery perfusion for 24 48, 72, or 96 h, followed by anti-Thy-1 Ab injection. The efficiency of transferring the plasmids into glomeruli was determined by observing GFP expression (supplemental Fig. 1D). Besides, the IRF-1 expression in the renal tissue was examined using Western blot, and the result showed that IRF-1 shRNA pretreatment for 72 h before giving anti-Thy-1 Ab could markedly silence IRF-1 expression induced by anti-Thy-1 Ab injection for 3 h (supplemental Fig. 1E).

To examine the roles of IRF-1 or caspases in mediating GMC apoptosis of Thy-1N rats, other normal male SD rats (160–200 g) were divided into four groups (n = 5), namely 1) NS, 2) Thy-1N, 3) control shRNA + Thy-1N, and 4) IRF-1 shRNA + Thy-1N, and the rats in the NS and Thy-1N groups were injected with the corresponding sera as mentioned above, but the rats allocated to control shRNA + Thy-1N and IRF-1 shRNA + Thy-1N were treated by the same method described previously (14). Furthermore, other normal SD rats (160–200 g) were again divided into six groups (n = 5): 1) control shRNA + Thy-1N; 2) IRF-1 shRNA + Thy-1N; 3) Z-IETD-FMK (caspase 8 inhibitor) + Thy-1N; 4) Z-DEVD-FMK (caspase 3 inhibitor) + Thy-1N; 5) DMSO + Thy-1N; and 6) NS. The rats allocated to Z-IETD-FMK + Thy-1N and Z-DEVD-FMK + Thy-1N were given the caspase inhibitors at a final concentration of 1 μm blood volume by intravenous injection and then treated with anti-Thy Ab (0.75 ml/100 g body weight). The renal cortexes of rats were collected at 3 h and day 7 by biopsies or sacrifice, the samples were embedded in paraffin or Epon 812, and the GMC changes were determined by TUNEL, electron microscopy, and light microscopy.

Microarray Analysis

Total RNA was extracted from GMC (6 wells) incubated with sublytic C5b-9 or renal cortices of Thy-1N rats (6 rats) for 40 min and 3 h using an RNeasy minicolumn (11). Biotinlabeled cRNA prepared from template cDNA was fragmented and hybridized to Affymetrix RAE230A arrays (done in triplicate). The arrays were biotin-labeled prior to scanning with a confocal scanner. Only those genes with a p value of ≤0.01 were included in the subsequent analysis. The expression data for each time point were compared with control, and a signal log ratio of 1 or greater was taken to identify genes with significantly different regulation.

Real-time PCR

The total RNA from cultured GMC or renal tissues was extracted, and IRF-1 mRNA was measured using real-time PCR. 1) To detect IRF-1, a fluorescence-labeled probe was used (forward primer, 5′-GTACAACTTGCAGGTGTCGC-3′; reverse primer, 5′-GCTGCCACTCAGACTGTTCA-3′) as well as a FAM/TAMRA-labeled probe, 5′-CCTCCACCTCTGAAGCTGCAACA-3′). 2) β-Actin was quantified using forward primer (5′-TCACCCACACTGTGCCCATCTATGA-3′), reverse primer (5′-CATCGGAACCGCTCATTGCCGATAG-3′), and FAM/TAMRA-labeled probe (5′-ACGCGCTCCCCCATGCCATCCTGCGT-3′). -Fold change was calculated with the relative C_T method as 2 raised to (normalized C_T in control sample − normalized C_T in stimulated sample).

Western Blot

The lysates of cultured GMC or extracts of renal cortices were prepared. Equal amounts of protein were subjected to electrophoresis on 12% SDS-PAGE, and the electroblotted membranes were then incubated with the primary antibodies to IRF-1, procaspase/cleaved caspase 9, procaspase/cleaved caspase 8, and procaspase/cleaved caspase 3, followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Renal Histological Examination

Paraffin sections were stained with H&E. Numbers of glomerular cells were quantified from counts of positive-stained nuclei and performed in a double-blinded manner by two independent observers counting under light microscopy. Ultrastructural changes were examined by transmission electron microscopy (14).

TUNEL Analysis

Tissue sections were treated with proteinase K and incubated with a 50-μl reaction mixture of terminal deoxynucleotidyltransferase for 60 min. The number of TUNEL-positive nuclei in 100 glomerular cross-sections was counted in a double-blinded manner under fluorescence microscopy (14).

Flow Cytometry

5 × 10⁵ GMC were resuspended in binding buffer containing Annexin V-APC and propidium iodide. The samples were analyzed on a FACScan flow cytometer. The percentage of apoptotic cells in a 10,000-cell cohort was determined by flow cytometry.

Luciferase Reporter Assay

To analyze IRF-1 and caspase 8 promoter activity, 2 × 10⁵ GMC were transfected with the rat 1.7-kb IRF-1 promoter or the rat 1.24-kb caspase 8 promoter (nt −1136 to +101) accompanied by pRL-SV40 as a transfection efficiency control. After 48 h, the cells were treated with sublytic C5b-9, anti-Thy-1 Ab + C6DS, anti-Thy-1 Ab + HIS, anti-Thy-1 Ab, and MEM for 40 min and 3 h. The luciferase activity was assayed in a TD20/20 luminometer. Moreover, part of the cells transfected with full-length caspase 8 promoter (nt −1136 to +101) or different promoter deletion fragments (nt −936 to +101, −736 to +101, −336 to +101, or −136 to +101) for 48 h was attacked by sublytic C5b-9 for another 3 h, and then the luciferase activity was detected.

In order to further assess the effects of IRF-1 overexpression or knockdown on caspase 8 promoter activity, GMC were first co-transfected with IRF-1 expression plasmids (pIRF-1) or IRF-1 shRNA and caspase 8 promoter plasmids for 48 h. The cells were subsequently divided into six groups, namely sublytic C5b-9, IRF-1 shRNA + sublytic C5b-9, control shRNA + sublytic C5b-9, MEM, pIRF-1, and pcDNA3.1, and the luciferase activity of each group was measured. Additionally, to further identify which caspase 8 promoter deletion fragments can be activated by IRF-1 overexpression, GMC were co-transfected with pIRF-1 and caspase 8 promoter plasmids of full-length or deletion fragments for 48 h, and luciferase activity was also examined.

Chromatin immunoprecipitation (ChIP)

DNA-binding proteins were cross-linked to DNA and lysed in SDS lysis buffer. DNA was sheared to 200–500-bp fragments by sonication for 30 s. The chromatin solution was precleared with salmon DNA/protein A-agarose 50% slurry for 30 min, and the supernatant was incubated with anti-IRF-1 antibody overnight. A proximal region in the caspase 8 promoter (nt −336 to −136) was amplified from the immunoprecipitated chromatin by PCR using four pairs of primers: primer 1 (sense, 5′-CTGTGACCAGTGGCTTTC-3′; antisense, 5′-CCAGACACTTTAGAGGGA-3′) (nt −235 to −122); primer 2 (sense, 5′-CCAGGAGGTGACTCTGTG-3′; antisense, 5′-GAGGGAAGAAATGGAGAA-3′) (nt −248 to −134); primer 3 (sense, 5′-CAGCGTCACCAACAATCC-3′; antisense, 5′-GGTCACAGAGTCACCTCC-3′) (nt −411 to −228); primer 4 (sense, 5′-CACCAACAATCCGTTCTA-3′; antisense, 5′-GGTCACAGAGTCACCTCC-3′) (nt −405 to −228).

Urine Protein Detection

The urinary samples in rats were collected at 24 h and day 7. The total protein contents in urine were measured by the total protein UC FS (DiaSys Diagnostic Systems, Holzheim, Germany).

Statistical Analysis

All data are given as mean ± S.D. The statistical significance (defined as p < 0.05) of the groups was evaluated by one-way analysis of variance with simultaneous multiple comparisons between groups by the Bonferroni method. All measurements of each sample were performed in triplicate.

Comparison of Gene Expression Profiles both in GMC following Sublytic C5b-9 Stimulation and in Renal Tissues of Rats with Thy-1N

Among 15,923 gene sequences expressed on the Affymetrix RAE230A oligonucleotide microarray, 928 genes and 979 genes were disturbed in the cultured GMC following sublytic C5b-9 stimulation (in vitro) at 40 min and 3 h, respectively, in which 536 and 486 genes were up-regulated at 40 min and 3 h, respectively. Moreover, 681 genes and 749 genes of them were perturbed at 40 min or 3 h after Thy-1N induction, and 667 genes as well as 173 genes were up-regulated at 40 min or 3 h from the renal tissue of Thy-1N rats (in vivo). Supplemental Table 1, A–D, highlights in part the genes whose mRNA levels were increased (i.e. related to signal transduction, apoptosis, extracellular matrix, early response, inflammation, etc.).

Among all differentially expressed genes, IRF-1, ATF3 (activating transcription factor 3), egr-1 (early growth response 1), C/EBPβ (CCAAT/enhancer-binding protein β), gadd45 (growth arrest and DNA-damage-inducible 45), CTGF (connective tissue growth factor), IL-6 (interlukin 6), IL-1β (interlukin 1β), HO-1 (heme oxygenase 1), schlafen 3, and cyclin L1 were found to be co-up-regulated both in vitro and in vivo (supplemental Table 1, A–D), indicating that these genes might be critically involved in Thy-1N pathogenesis.

Up-regulation of IRF-1 Expression both in GMC Induced by Sublytic C5b-9 Attack and in Renal Tissues of Rats with Thy-1N

Of the co-up-regulated genes, IRF-1 was selected to further study its function because several experiments have reported that IRF-1 is associated with tumor cell apoptosis (20, 22). Thus, IRF-1 expression levels both in the GMC attacked by sublytic C5b-9 and in the renal tissues of Thy-1N rats were determined. As expected, the data showed that IRF-1 mRNA and protein both in vitro and in vivo were increased at 20 min, peaked at 3 h, and then reduced at 6 and 12 h (supplemental Fig. 2, A–D). In order to avoid the effects of MEM, Thy-1 Ab, or human serum, and to determine that C6 is necessary for C5b-9 assembly on GMC membrane, cultured GMC were also treated with sublytic C5b-9, Thy-1 Ab + C6DS, Thy-1 Ab + HIS, Thy-1 Ab, and MEM for 3 h. The results demonstrated that IRF-1 expression was up-regulated markedly after sublytic C5b-9 exposure (Fig. 1, A and B). Furthermore, IRF-1 expression increased at 3 h in the renal tissues of Thy-1N rats (Fig. 1, C and D). These findings confirmed that the GMC attacked by sublytic C5b-9 and renal tissue of Thy-1N rats displayed increased IRF-1 expression.

Activation of Caspase 9, Caspase 8, and Caspase 3 both in GMC Induced by Sublytic C5b-9 Attack and in Renal Tissues of Rats with Thy-1N

To ascertain whether sublytic C5b-9 attack can activate caspase, the expression of procaspases 9, 8, and 3 and cleaved caspases 9, 8, and 3 both in the GMC upon sublytic C5b-9 attack and in the renal tissues of Thy-1N rats was determined. The results suggested that procaspase 8 and cleaved caspases 8 and 3 increased significantly at 40 or 80 min (cleaved caspase 9 emerged at 3 h) and peaked at 3 h in vitro. Procaspase 8 and cleaved caspases 9, 8, and 3 began to increase at 40 min, 80 min, or 3 h, peaking at 3 h in vivo (supplemental Fig. 2, E and F). In addition, the results from different groups showed that the protein levels of cleaved caspases 9, 8, and 3 were up-regulated significantly in the GMC at 3 h after sublytic C5b-9 incubation (Fig. 2A) and in the renal tissues of Thy-1N rats (Fig. 2B), indicating that sublytic C5b-9 induced caspase pathway activation in the GMC in Thy-1N rats.

Effects of IRF-1 Overexpression or Knockdown on GMC Apoptosis Induced by Sublytic C5b-9 Stimulation

To confirm the effect of IRF-1 on the GMC apoptosis in response to sublytic C5b-9, the rate of GMC apoptosis mediated by sublytic C5b-9 was detected, and the result demonstrated that sublytic C5b-9 could increase GMC apoptosis (supplemental Fig. 3A). Afterward, the plasmids of pcDNA3.1 expressing IRF-1 (pIRF-1) or pcDNA3.1 (vector) and IRF-1 shRNA were transfected into GMC for 48 h, respectively, and then with or without sublytic C5b-9 stimulation for 3 h. In fact, the cultured GMC were divided into the following treatments: 1) MEM, pcDNA3.1, sublytic C5b-9, pcDNA3.1 + sublytic C5b-9, pIRF-1, and pIRF-1 + sublytic C5b-9 and 2) MEM, sublytic C5b-9, control shRNA + sublytic C5b-9, and IRF-1 shRNA + sublytic C5b-9. The results revealed that IRF-1 expression in the sublytic C5b-9, pcDNA3.1 + sublytic C5b-9, pIRF-1 and pIRF-1 + sublytic C5b-9 groups was greatly enhanced, especially much more in the pIRF-1 + sublytic C5b-9 group. In contrast, IRF-1 expression decreased in the IRF-1 shRNA + sublytic C5b-9 group (supplemental Fig. 3, B and C). In addition, GMC apoptosis in the pIRF-1 + sublytic C5b-9 group or in the IRF-1 shRNA + sublytic C5b-9 group exhibited a significant increase or decrease, respectively (Fig. 3A), providing evidence that IRF-1 plays a role in the GMC apoptosis triggered by sublytic C5b-9 attack.

Changes of Caspase 9, 8, and 3 Activation by IRF-1 Gene Knockdown in GMC Stimulated by Sublytic C5b-9

To determine whether GMC apoptosis induced by sublytic C5b-9 can be caused by IRF-1 activation of caspases, the cultured GMC were again divided into MEM, sublytic C5b-9, control shRNA + sublytic C5b-9, and IRF-1 shRNA + sublytic C5b-9 groups. Then activation of caspases 9, 8, and 3 in the GMC transfected with IRF-1 shRNA for 48 h followed by sublytic C5b-9 treatment for another 3 h was examined by measuring the corresponding cleavages. As presented in Fig. 3B, cleaved caspases 8 and 3 in the IRF-1 shRNA + sublytic C5b-9 group were significantly lower than that in the sublytic C5b-9 and control shRNA + sublytic C5b-9 groups, but the cleaved caspase 9 did not decrease in the same group, implying that IRF-1 gene knockdown had an inhibiting effect on the activation of caspases 8 and 3 induced by sublytic C5b-9. These data implicated that IRF-1-promoted GMC apoptosis was related to caspase 8 activation.

Effects of Inhibiting Caspase 8 and 3 Activation on GMC Apoptosis Mediated by Sublytic C5b-9 or Induced by IRF-1 Overexpression

To further demonstrate the roles of caspase 8 and 3 activation in GMC apoptosis mediated by sublytic C5b-9 or induced by IRF-1 overexpression, the GMC were divided into six groups with the following treatments: DMSO + sublytic C5b-9, Z-IETD-FMK + sublytic C5b-9, Z-DEVD-FMK + sublytic C5b-9, DMSO + pIRF-1, Z-IETD-FMK + pIRF-1, and Z-DEVD-FMK + pIRF-1. GMC apoptosis was evaluated at 3 h (here the method and time of pIRF-1 transfected into GMC was the same as previously mentioned). The results indicated that the relative amount of GMC apoptosis induced by sublytic C5b-9 or IRF-1 overexpression could be significantly reduced by using caspase 8 or 3 inhibitor, compared with the DMSO + sublytic C5b-9 or DMSO + pIRF-1 group (Fig. 3C), suggesting that inhibiting caspase 8 or 3 activation could actually decrease GMC apoptosis in response to sublytic C5b-9 stimulation or IRF-1 overexpression.

Increase of IRF-1 and Caspase 8 Promoter Activity in GMC Induced by Sublytic C5b-9 Stimulation

In order to further clarify whether the IRF-1 or caspase 8 gene was directly activated by sublytic C5b-9 attack, the activity of the IRF-1 and caspase 8 promoters was analyzed. As shown in Fig. 4 A, the luciferase activity of the 1.7-kb IRF-1 promoter increased in the GMC exposed to sublytic C5b-9 for 40 min (1.95-fold versus MEM) and 3 h (2.61-fold versus MEM). Moreover, as shown in Fig. 4B, the luciferase activity of the 1.24-kb caspase 8 promoter was elevated at 40 min (1.56-fold versus MEM) and markedly increased in the GMC at 3 h after sublytic C5b-9 stimulation (2.24-fold versus MEM). These results were in agreement with the findings that sublytic C5b-9 up-regulated IRF-1 and caspase 8 expressions in vitro, providing evidence that IRF-1 and caspase 8 promoter activity in the GMC incubated with sublytic C5b-9 can be directly up-regulated.

To further decide which element was involved in caspase 8 gene activation upon sublytic C5b-9 attack, caspase 8 promoter fragments were generated, and the promoter activities of the GMC transfected with these constructs for 48 h and attacked by sublytic C5b-9 again for 3 h were detected. The data presented in Fig. 4C suggested that only the activity of the caspase 8 promoter deletion fragment (nt −136 to +101) was notably less than that of the full-length caspase 8 promoter (nt −1136 to +101) or other deletion fragments (nt −936 to +101, −736 to +101, and −336 to +101), indicating that a key element in response to sublytic C5b-9 stimulation might be located within the nt −336 to −136 proximal region of the caspase 8 promoter.

Regulation of Rat Caspase 8 Promoter Activity in GMC by IRF-1 Gene Knockdown or Overexpression in GMC Exposed to Sublytic C5b-9 Stimulation

To test whether IRF-1 knockdown or overexpression can affect caspase 8 gene transcription triggered by sublytic C5b-9 attack, the GMC were transfected with the 1.24-kb caspase 8 promoter and IRF-1 shRNA or pIRF-1 plasmids for 48 h, and then the GMC was treated with or without sublytic C5b-9 for another 3 h. The caspase 8 promoter activity in the six groups (sublytic C5b-9, IRF-1 shRNA + sublytic C5b-9, control shRNA + sublytic C5b-9, MEM, pIRF-1, and pcDNA3.1) was measured, and data suggested that caspase 8 promoter activity was significantly decreased in the IRF-1 shRNA + sublytic C5b-9 group and markedly increased in the pIRF-1 group (Fig. 5A), indicating that IRF-1 expression was involved in the elevation of caspase 8 promoter activity in the GMC triggered by sublytic C5b-9. In addition, to identify the elements required for caspase 8 promoter activity in response to IRF-1, the GMC were transfected with the luciferase reporter constructs containing the 1.24-kb caspase 8 promoter (nt −1136 to +101) or different promoter deletion fragments (nt −936 to +101, −736 to +101, −336 to +101, and −136 to +101) and pIRF-1 (overexpression plasmid) for 48 h and then with sublytic C5b-9 attack for 3 h, and caspase 8 promoter activity was again determined. As presented in Fig. 5B, only the caspase 8 promoter activity (nt −136 to +101) + pIRF-1 group showed an effective decrease relative to the full-length caspase 8 promoter + pIRF-1 or other deletion fragments + pIRF-1 group, suggesting a critical element in the nt −336 to −136 region at the 5′-end of caspase 8 exon-1 for IRF-1.

Identification of IRF-1 Binding to Rat Caspase 8 Promoter Element in GMC Induced by Sublytic C5b-9 Attack or by IRF-1 Overexpression

In order to further reveal that IRF-1 can bind to the region of nt −336 to −136 of the caspase 8 promoter, GMC were first divided into the following treatment groups for 3 h: sublytic C5b-9, anti-Thy-1 Ab + C6DS, anti-Thy-1 Ab + HIS, anti-Thy-1 Ab, and MEM. Then a ChIP assay was performed using anti-IRF-1 antibody or control IgG, and immunoprecipitated DNA was amplified using four different pairs of primers for the proximal promoter regions of the caspase 8 gene. The positive result was obtained only by using primer 3 (nt −411 to −228), indicating that IRF-1 induced by sublytic C5b-9 could bind to the element (nt −411 to −228) of rat caspase 8 promoter in the GMC upon sublytic C5b-9 attack (Fig. 6A). Meanwhile, the negative PCR result was achieved by using primers 1, 2, and 4 (data not shown). Furthermore, the cultured GMC were transfected with pIRF-1 or pcDNA3.1 plasmids for 48 h, and a ChIP experiment was also performed. The immunoprecipitated DNA was again amplified by PCR using the above mentioned four pairs of primers, resulting in a positive result from primer 3 (Fig. 6B) and negative results from the other three pairs of primers (data also not shown). Collectively, the findings manifested that overexpressed IRF-1 could bind to the element (nt −411 to −228) of caspase 8 promoter (Fig. 6B), indicating that up-regulation of rat caspase 8 promoter activity in response to sublytic C5b-9 attack was due to increasing IRF-1 binding to the caspase 8 promoter element.

It is worth mentioning that the potential transcription factor-binding sites in the promoter of rat caspase 8 gene (nt −336 to −228) were predicted using the computer program TFSEARCH (version 1.3), and all of the possible elements are underlined in supplemental Fig. 4, including elements for binding of heat shock factor, caudal type homeobox A, CAP1 (adenylate cyclase-associated protein 1), EVI-1 (ecotropic viral integration site 1), MZF1 (myeloid zinc finger 1), IK-2 (IκB kinase-like 2), YY1 (Yin and Yang 1 protein), GATA-1 (GATA-binding factor 1), NIT2 (nitrilase 2), GCN4 (general control non-repressible 4), and AP-1 (activator protein 1) but not the IRF-1-binding site. However, the ChIP assay suggested that the IRF-1 protein could bind to this element of rat caspase 8 promoter in the GMC upon sublytic C5b-9 attack (Fig. 6A).

Effects of IRF-1 Gene Knockdown on Caspase 8 and Caspase 3 Expression in Renal Tissue of Rats with Thy-1N

To demonstrate the role of IRF-1 in promoting GMC apoptosis in vivo, normal SD rats were classified into four groups: 1) NS, 2) Thy-1N, 3) control shRNA + Thy-1N, and 4) IRF-1 shRNA + Thy-1N. The expression of renal IRF-1, caspase 8, and caspase 3 protein in the four groups at 3 h after nephritis establishment was examined, and the results showed that expression of IRF-1 (Fig. 7A) and cleaved caspases 8 and 3 (Fig. 7B) in the IRF-1 shRNA + Thy-1N group was markedly down-regulated as compared with the Thy-1N or control shRNA + Thy-1N group, although the protein levels of IRF-1 and cleaved caspases 8 and 3 in the IRF-1 shRNA + Thy-1N group were higher than in the NS group. Taken together, the data suggest that renal IRF-1 gene knockdown could inhibit renal caspase 8 and 3 activation of Thy-1N rats.

Effects of IRF-1 Knockdown or Caspase 8 and 3 Inhibition on GMC Apoptosis and Other Pathologic Changes, Including Urinary Protein of Rats with Thy-1N

To further evaluate the role of IRF-1 in promoting GMC apoptosis of Thy-1N rats, normal SD rats were divided into six groups: 1) control shRNA + Thy-1N, 2) IRF-1 shRNA + Thy-1N, 3) Z-ITED-FMK + Thy-1N, 4) Z-DEVD-FMK + Thy-1N, 5) DMSO + Thy-1N, and 6) normal serum (NS). The numbers of TUNEL-positive cells and glomerular cells in renal tissues at 3 h and 7 days were observed. The results revealed that the numbers of TUNEL-positive cells were substantially decreased in IRF-1 shRNA + Thy-1N or Z-ITED-FMK + Thy-1N and Z-DEVD-FMK + Thy-1N groups (Fig. 8A), and under electron microscopy, the apoptotic changes, including irregular aggregation of chromatin at the periphery of the nucleus and condensation of the nuclear chromatin in IRF-1 shRNA + Thy-1N, Z-ITED-FMK + Thy-1N, and Z-DEVD-FMK + Thy-1N groups, were reduced (Fig. 8B). Meanwhile, the numbers of glomerular cells in the above mentioned groups on day 7 under light microscopy were also significantly less than the number in control groups, although the numbers were still higher than that in the NS group (Fig. 8C). Additionally, urinary protein content (mg/24 h) at 24 h and on day 7 was also significantly less than that in control groups (Fig. 8D), implicating that IRF-1 gene silencing or caspase 8 and 3 inhibition could alleviate GMC apoptosis, secondary proliferation, and urinary protein in the rats with Thy-1N.