Complement component C5a induces aberrant epigenetic modifications in renal tubular epithelial cells accelerating senescence by Wnt4/βcatenin signaling after ischemia/reperfusion injury

Results

C5a induces DNA methylation changes in RTECs

To study whether Complement could modulate DNA methylation in renal cells, we stimulated human RTECs with C5a anaphylatoxin for 24 hours and performed a whole-genome DNA methylation study. Methylation analysis was performed for single CpGs and also for sets of predefined genomic regions such as CpG islands, promoters and tiling regions (5-kb spanning regions). In untreated cells (indicated as basal), the highest number of methylated tiling regions were found in chromosome 1, 12, 10, 7 and 3. They cover the 50% of the total tiling regions, whereas chromosomes 4, 18 and 21 contained the fewest methylated regions (Figure 1A). C5a stimulation decreased the DNA methylation in the overall genome when we consider the single CpG sites (Figure 1B). In particular, considering the tiling regions, most DNA regions were hypomethylated and located in specific chromosomes (Figure 1C, Supplementary Table 1).

Figure 1. C5a-associated changes in DNA methylation as indicated by whole-genome bisulfite assay in RTEC. (A) Chart showing the number and the frequency of methylated regions (tiling regions) identified in RTEC at basal level. The left vertical axis represents the number of methylated regions per each chromosome. The right vertical axis indicates the cumulative percentage of the total number of occurrences. The red concave curve is the cumulative function indicating that the 50% of the total methylated regions in RTEC are covered by chromosomes 1, 12, 10, 7 and 3. (B) DNA methylation levels at the single CpG sites for RTEC at basal level (blue line) and for RTEC stimulated by C5a (orange line). C5a decreased the DNA methylation in the overall genome. (C) Graph showing the mean DNA methylation levels of tiling regions, shared for chromosomes, in RTEC (blue line) and in C5a-stimulated RTEC (red line). The central axis shows the mean β methylation value. (D) Scatterplot of the CpG site methylation comparison, colored according to the combined ranks of a given site. Values are represented as mean differences (mean.diff) between stimulated and unstimulated RTEC for each CpG site. (Combined rank: difference in mean methylation levels of stimulated and non-stimulated RTEC, the quotient in mean methylation and the t test are ranked for all regions. This value aggregates them using the maximum, i.e. worst rank of a site among the three measures.)

We then performed differential methylation analysis according to the combined Rank parameter and found 74 hypermethylated and 320 hypomethylated CpG sites in RTEC stimulated with C5a (Figure 1D). Next, after the differential methylation analysis of the tiling regions we selected the best 150 combined ranks having a threshold of Δβ>0.05. We found 142 regions, 88 hypomethylated and 54 hypermethylated (Supplementary Table 2). In addition, when we analyzed differentially methylated CpG island, we found only 3 island hypermethylated and 8 island hypomethylated by C5a (Supplementary Figure 4F).

In order to gain insight into the biological mechanisms involved in the response to C5a, we performed a pathway and a gene ontology (GO) analysis of genes spanned in the identified DNA regions (Supplementary Figure 1). The pathway analysis of regions hypo- and hypermethylated by C5a showed the involvement of biological processes as “Aryl Hydrocarbon Receptor Signaling”, “cell cycle: G2/M DNA damage checkpoint regulation” and “Wnt/βcatenin pathway” (p=0.019, 0.027 and p=0.033, respectively). In particular, we found three hypomethylated genes as central node of the networks: BCL9, CYP1B1 and CDK6 (Supplementary Figure 2). These genes are involved in the cell cycle progression and apoptosis. In addition, also the GO analysis showed an enrichment of DNA regions containing genes involved in the cell cycle checkpoint (Supplementary Figure 3).

We then validated the methylation status of the three genes by pyrosequencing (Figure 2A, 2B). We confirmed that BCL9, CYP1B1 and CDK6 genes were hypomethylated by C5a (p<0.05) with mean differences in methylation levels of about 8-10% (Figure 2A, 2B). In particular, the CYP1B1 region contained multiple CpG sites most of which were concordantly hypomethylated.

Figure 2. Analysis of methylation levels for BCL9, CYP1B1 and CDK6 in different lots of C5a-stimulated RTEC compared to the respective basal conditions. Pyrosequencing assays were performed on the same regions that we found to be methylated in the whole-genome assay. The pyrosequencing assays confirmed that the DNA of these regions were differentially methylated in C5a-stimulated RTEC (B panel) compared to basal condition (A panel). Representative pyrograms show methylation levels of the indicated CpG sites in the gene promoter.

Overall, these data demonstrated the functional effect of the C5a anaphylatoxin to re-program in-depth the tubular cells epigenetic state, promoting a global hypomethylation in different chromosomes. In addition, the DNA methylation changes were consistent with cell cycle pathway.

C5a induced DNA methylation regulating BCL9, CYP1B1 and CDK6 gene expression

To confirm the critical role of DNA methylation in regulating the BCL9, CYP1B1 and CDK6, we assessed the gene expression from C5a-stimulated RTEC compared with untreated cells (Figure 3). The genes were differentially expressed in C5a-stimulated RTEC; their expression was inversely associated with the relative DNA methylation (Figure 3D–3F): BCL9, CYP1B1 and CDK6 genes were hypomethylated with a significant up-regulation of their gene expression. (Figure 3A–3C: mean fold changes of BLC9 2.19±0.5, CYP1B1 2.38±0.59 and CDK6 2.49±0.62, *p<0.05, **p<0.01).

Figure 3. BCL9, CYP1B1 and CDK6 gene expression is regulated by the DNA methylation. (A–C) Gene expression of BCL9, CYP1B1 and CDK6 evaluated by qRT-PCR in the C5a stimulated-RTEC and compared to normal RTEC cultured for 24h. The gene expression validated the genes analyzed for the methylation using the pyrosequencing assay. Expression levels were significantly different in C5a-stimulated RTEC compared with normal cells. Gene expression levels were normalized to the housekeeping gene GAPDH. (D–F) BCL9, CYP1B1 and CDK6 methylation levels of C5a-stimulated RTEC compared to basal condition. Results are means±SD, n=3. (G–I) Gene expression of BCL9, CYP1B1 and CDK6 of C5a stimulated-RTEC not treated or treated with 1 μM 5-aza-2’-deoxycytidine. The DNA demethylation agent reduced the methylation levels in the promoters of the three genes. The DNA methylation status of these three DNA regions was determined by qMSP real-time analysis. The degree of fully methylated molecules at a specific locus was expressed as a PMR index. The percentage PMR was calculated as described in the Materials and methods. Qiagen methylation control DNA was used as full methylated reference. (J–L) Gene expression of BCL9, CYP1B1 and CDK6 of C5a stimulated-RTEC not treated or treated with 1 μM aza. Results are means±SD, n = 3.*p<0.05.

Next, to further validate the regulatory involvement of DNA methylation in BCL9, CYP1B1 and CDK6 gene expression, we treated RTEC for 24h with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza, 1μM), a DNA hypomethylating agent. Following 5-aza treatment, we assessed BCL9, CYP1B1 and CDK6 hypomethylation (Figure 3G, 3H, 3I). Effectively, after 5-aza induced-DNA demethylation, we found that the mRNA expression of BCL9, CYP1B1 and CDK6 by qPCR significantly increased (Figure 3J, 3K, 3L). These results indicated that gene expression of BCL9, CYP1B1 and CDK6 is directly regulated by DNA methylation and that C5a is an epigenetic mediator that caused hypomethylation of genes that are involved, at least in part, in the Wnt/βcatenin signaling.

Characterization of C5a-induced cellular senescence in RTEC

Because the three validated genes were clustered in Wnt/βcatenin pathway [22, 23] that is involved in aging processes [24], we tested the hypothesis that C5a might directly promote RTEC senescence in vitro. We then stimulated RTEC by C5a for 3h and 24h (Figure 4B and 4F, 4J) followed by culture in fresh medium for additional 48h (4B) or 24h (4J); interestingly, C5a stimulated-RTEC acquired a senescent phenotype as determined by significant increase in SA-βGAL positivity (Figure 4B, 4F, 4J). C5a exposed RTEC become larger (Figure 4F, arrow) and polynucleated (Figure 4B, arrow). The inhibition of receptor C5aR1 appeared to significantly protect tubular cells from C5a-induced senescence (Figure 4C, 4G and 4K), indicating that C5a effect was mediated by this receptor. RTEC exposed to H₂O₂ (Figure 4D, 4H and 4L) were used as positive control of cellular senescence. Quantification of senescent cells in Figure 4M showed that the induction of C5a mediated RTEC senescence was statistically significant in all the condition tested and also in presence of different time of activation (3h and 24h; *p<0.05, images at 3h+24 not showed).

Figure 4. SA-β Gal staining in RTEC treated with C5a with or without C5aR1 blocking. SA-β-Gal activity in early passage RTEC exposed to 100 nM C5a for 3h (B, F) or 24h (J). For C5aR1 inhibition, mouse monoclonal anti-C5aR1 was pre-incubated for 1h before the C5a exposure and then maintained in fresh medium for 24h or 48h (C, G, K). More SA-β-gal+ cells were observed after C5a exposure, senescent RTEC appeared enlarged and morphologically distinct from the normal cells at the same passage with formation of larger and polynucleated cells (B and F, arrows). Untreated cells are also named as Basal (A, E, I). H₂O₂ exposed cells were used as positive control of senescence (D, H, L). Representative images were acquired by phase contrast microscopy. (M) Quantification of SA-β-Gal+ cells cultures. The ratio of cells positive for SA-β-gal activity was calculated by examining five not overlapping fields per condition (6-well plate). The results are presented as the mean ± SD of three independent experiments (*p< 0.05), Magnification 40X.

Since cellular senescence is characterized by cell cycle arrest, we performed the MTT assay to assess the number of viable RTEC (Figure 5A). C5a led to a reduction in cellular proliferation compared to untreated cells (MTT C5a 24h: 0.134±0.03 vs basal 24h: 0.219±0.02); the inhibition of proliferation was stable even in presence of short C5a stimulation with 48h of culture in fresh medium (C5a 3h+48h: 0.013±0.04 vs basal). RTEC exposed to H₂O₂ were used as internal control showing a 54% inhibition of proliferation. In addition, cells counting performed by Trypan blue from the supernatants of cell culture did not indicate significant variations in the viable/dead cells number ratio (data not shown).

Figure 5. Altered cell proliferation and p53 expression after C5a exposure in RTEC. (A) MTT assay of RTEC in logarithmic growth phase treated with the same dosage of C5a showed an inhibition of cell proliferation. (B) TP53 gene expression level in C5a stimulated RTEC. Data are expressed as the mean ± SD (*p< 0.05 versus basal condition).

Next, we tested the activation of p53 /TP53 gene pathway that is crucial in regulating premature senescence and cell- cycle arrest [25]; therefore, we performed a cell cycle assay and assessed the expression of TP53. Three hours after C5a exposure, we observed a significant increase in p53 transcript that further increased after 24h and 48h of culture (mean fold change: C5a 3h+48h: 5.6±1.64, p=0.008; C5a 3h+24h: 3.17±1.32, p=0.004, versus basal 1±0.0). The p53 expression increase was higher even after a short C5a stimulation (3h) followed by 48h of normal culture in fresh medium (Figure 5B); effectively most of cells were halted in G0/G1 phase of the cell cycle (83% versus 58% of non-stimulated cells), indicating that C5a affected the arrest of the cell cycle (Supplementary Figure 4A).

C5a induced the development of Secretory Associated Senescence Phenotype (SASP) in RTEC

After p53 activation, cells transiently express p21Cip1/Waf1 [26] a Cyclin-Dependent Kinase Inhibitor (CKI) that can lead to a chronic state of senescence, triggering G1 cell cycle arrest or leading to apoptosis. Therefore, we measured the p21 protein level in C5a stimulated RTEC and we found a significant protein increase after both short time (3h) and longer time (24h) of C5a exposure. (Figure 6A, 6B, p21 C5a 3h+24: 1.45±0.17; p<0.01, C5a 24h+24: 1.28±0.37, p<0.05). In particular, this effect was more evident after 24h of C5a exposure followed by 72h of normal culture. Since p21 could transiently increase after an injury, we assessed also the p16INK4a protein level. Stimulation with C5a significantly induced a constant augment in protein expression of p16INK4a compared to untreated condition (basal) (Figure 6A and 6B).

Figure 6. Cell cycle negative regulators expression levels and SASP pro-inflammatory cytokines transcripts are up regulated by C5a. (A–B) Representative p21 Waf1/Cip1 and p16INK4a western blot of C5a-stimulated RTEC and quantification. Protein expression was normalized to βactin. (C–I) Time course of IL-6, MCP1, CTGF, SERPIN1 (PAI-1), BCL9, CYP1B1 and CDK6 gene expression in RTEC stimulated for 3h or 24h by C5a followed or not by 24h or 48h of normal culture was assessed by qPCR. Data were normalized to GAPDH. *p<0.05, **p<0,01, n=3. H₂O₂ (100-300 μm) was used as positive control of senescence.

Subsequently, we investigated the gene expression of IL-6, MCP-1, CTGF and PAI-1, that are chemokines typical associated with the Senescence-Associated Secretory Phenotype (SASP) [27]. As observed for p21 protein, we found a significant increase in IL-6, SERPINE1 (PAI-1), CCL2 (MCP-1) and CCN2 (CTGF) after three hours of C5a exposure followed by 24h of normal culture (Figure 6C–6F; mean fold change IL-6: 8.05±0.24; PAI-1: 6.58±1.72 versus baseline, p<0.01). Interestingly, IL-6, PAI-1 and CTGF gene expression levels remained high even after 48h of culture from C5a stimulation. Significant expression increases for all these genes, except MCP1, was found also after 24h of C5a exposure followed by 24h of normal culture. Increase in the gene expression of each pro-inflammatory cytokine was found also by 300 μM H₂O₂, that is a stressor inducer of SASP (Figure 6C–6F).

Finally, in order to gain insight in BCL9, CYP1B1 and CDK6 genes expression we performed qPCR at different time points. All genes showed a significant up-regulation both after a short C5a stimulation (3h) followed by 24 or 48h of culture in normal medium and after 24h of C5a stimulation followed by 24h of culture. A similar increase was detected after 300 μM H₂O₂ exposure (Figure 6G–6I).

Taken together these results demonstrate that C5a can induce tubular senescence in vitro, characterized by morphological and functional changes such as growth arrest and acquirement of a pro-inflammatory phenotype named SASP.

C1-INH modulated Wnt pathway activation during I/R leads counteracting renal senescence

We further investigated whether I/R might promote the development of tubular senescence in vivo. To this aim, we used a swine model of renal I/R injury in which pigs were treated with C1-Inhibitor to modulate Complement activation [28, 29]. We first investigated Wnt4 expression on renal sections obtained at 24 hours after I/R injury since the in vitro data showed an aberrant methylation and increased expression of FZD6 (Supplementary Figure 4B, 4D), a well-known Wnt4 receptor [30].

During I/R injury, the protein level of Wnt4 increased compared to T0 (Figure 7A). The expression mainly localized to the apical side (Figure 7A, T24 CTRL arrow) of epithelial tubular cells and was more evident in area of tubular damage characterized by vacuolization and interstitial edema. Moreover, Wnt4 expression was significantly reduced by treatment with C1 inhibitor (Figure 7A, T24 C1-INH). Subsequently, we assessed the Wnt/β-catenin pathway activation by measuring the level of β-catenin activation. One day after I/R injury, an increased number of β-catenin⁺ cells were detected (Figure 7B). Compared with the T0, β- catenin protein was up regulated predominantly in renal tubules of the I/R injured kidney. β-catenin was localized in the cytoplasm and the nuclei of tubular epithelial cells. (Figure 7B, T24 CTRL). After 24h from the reperfusion, relative β-catenin levels were increased compared to the T0, suggesting that induction of Wnt expression would result in an accumulation of β-catenin in injured and senescent kidney. Less β-catenin⁺ cells were present in C1 inhibitor-treated pigs (Figure 7B, T24 C1-INH), again showing the involvement of Complement in the activation of this pathway.

Figure 7. Wnt4/βcatenin pathway and inflammaging markers are activated in tubular cells after I/R and modulated by C1-INH treatment. (A–B) Immunoistochemical stainings showing the tubular Wnt4 and βcatenin increase after 1 day of I/R injury and the C1-INH-mediated modulation. IHC was performed on paraffin kidney sections. Arrows indicate positive tubular staining. (in the right) Graphical representation of Wnt4 and βcatenin protein expression level in the different groups. (n=5, p value as indicated, scale bar as indicated).(C) Representative SA-β Gal stained kidney tissues revealed an higher number of senescent cells after 24 h of I/R injury compared to T0. Treatment with C1-INH restored SA-β Gal at basal expression. Arrows indicate positive tubular staining on cryo tissues. (in the right) Graphical representation %SA-βGal area fraction. (n=5, p value as indicated, scale bar as indicated). (D) Representative micrographs indicating p16INK4a protein expression in different groups of swine, as indicated. Boxed areas are enlarged in the bottom of each micrographs. In the T0, p16INK4a had constitutive level and was localized in tubular nuclei (black arrow). Biopsies after 24h from reperfusion (T24 CTRL) showed increased nuclear (black arrow) and cytoplasmic staining (white dotted arrows). C1-INH restored p16INK4a at basal expression; limiting the cytoplasmic p16INK4a expression. (in the right) Graphical representation of p16INK4a area fraction in the different groups. (n=5, p value as indicated, scale bar as indicated).

To further clarify the final effect of the Wnt activation, we analyzed the tubular senescence by SA β-gal, p16 and p21 staining. As shown in Figures 7C and 7D and 8A (T24 CTRL), the Wnt/β-catenin activation was associated with SA β-gal positivity and increase in tubular p16 and p21 24h after I/R injury, Regards to p16 and p21, we detected beyond the constitutive nuclear staining (black arrow) (Figures 7D, 8A, T24 CTRL], a strong and diffuse cytoplasmic staining (white dotted arrow) that was described to have a negative clinical significance [31–33]. Furthermore, to assess the acquirement of SASP in vivo, we stained for IL-6 (Figure 8B) and found a statistically significant increase, as shown in Figure 8B and 8D.

Figure 8. p21 and IL-6 are induced in tubular cells after I/R and modulated by C1-INH treatment. (A) Representative micrographs indicating p21/CIP1 protein expression in different groups of swine after I/R injury, as indicated. Boxed areas are enlarged at the bottom of each micrographs. Compared to T0, biopsies after 24h from reperfusion (T24 CTRL) showed increased nuclear (black arrow) and cytoplasmic staining (white dotted arrow). C1-INH treatment restored p21 at basal expression; limiting the cytoplasmic p21 expression. (B) Representative micrographs show the expression and localization of IL-6, a marker of SASP in T0 (right), T24 CTRL (middle) and T24 C1-INH (right) groups. Arrows indicate positive tubular cells. Compared to T0, biopsies after 24h from reperfusion (T24 CTRL) showed increased IL-6 expression, predominantly at tubular cells. Treatment with C1-INH counteracts IL-6 increase. (C, D) Graphical representation of p21 and IL-6 area fraction in the different groups. (n=5, p value as indicated).

As shown in Figure 7A and 7B, Complement inhibition significantly abolished the Wnt/β-catenin activation induced by I/R. Consistently with the decreased activation of Wnt/β-catenin pathway by C1-inhibitor, the reduction of Wnt4 was associated to a block of SA-β gal enzymatic tubular activity (Figure 7C), to a reduction of p16 and p21 cytoplasmic expression (Figures 7D, 8A and 8C) and to a downregulation of IL-6 production. (Figure 8B and 8D). These results provide in vivo proof that Complement modulation can reduce tubular senescence by regulating Wnt/β-catenin pathway and inducing SASP in vivo.

Abbreviations

AhR: Aryl Hydrocarbon Receptor Signaling; AKI: Acute Kidney Injury; Bas: Basal condition; BCL9: B-Cell CLL/Lymphoma 9; C1-INH: C1-Inhibitor; CCDC6: Coiled-Coil Domain Containing 6; CKD: Chronic Kidney Disease; CTRL: Control group; CYP1B1: Cytochrome P450 Family 1 Subfamily B Member 1; DNMT: DNA methyl transferase; FDZ6: Frizzled Class Receptor 6; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; I/R: Ischemia/Reperfusion; RTEC: Renal Tubular Epithelial Cells; SASP: Senescence-Associated Secretory Phenotype; SA-βGAL: Senescence-Associated-βgalactosidase; SD: Standard Deviation; SERPINE1 (PAI-1): Serpin Family E Member 1 (Plasminogen Activator Inhibitor, Type I); TP53: Tumor Protein P53; qMSP: Quantitative Methylation Specific PCR; 5-aza: 5-aza-2’-deoxycytidine or Decitabine.

Author Contributions

Giuseppe Castellano, Rossana Franzin, Fabio Sallustio and Alessandra Stasi performed all the experiments and statistical analyses, Giuseppe De Palma performed MSP-qPCR validation analysis, Barbara Banelli and Massimo Romani performed the pyrosequencing assay and analysis, Giuseppe Lucarelli, Michele Battaglia, Antonio Crovace, Francesco Staffieri, Pasqual Ditonno performed the swine model of IRI, Chiara Divella collected and provided the pig samples, Giuseppe Grandaliano, Paolo Cravedi, Vincenzo Cantaluppi and Loreto Gesualdo supervised all the project. All authors were involved in data interpretation. Giuseppe Castellano and Rossana Franzin wrote the paper, all authors gave final approval of the submitted version.

Acknowledgments

This work was supported by University of Bari ‘‘Aldo Moro’’ and Ministero della Salute (Ricerca Finalizzata 2009, GR-2009-1608662, granted to G. C. and G. G.). The authors thank Angelica Intini and Angela Picerno for excellent technical assistance. The authors thank Beatris Oortwijn and Edwin van Amersfoort from Pharming Group NV, Leiden, the Netherlands for the unrestricted research grant supporting the study.

Conflicts of Interest

The authors of this manuscript have no conflicts of interest to disclose.

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