*3.5. Effect of HO-1 Depletion on Complement Component 3a Receptor (C3aR)*

C3aR staining was detected in both tubules and glomeruli of kidney tissue sections (Figure 5, Table 1). In glomeruli, C3aR immunolocalized predominantly in podocytes and was reduced in those of *Hmox1*−*/*<sup>−</sup> rats. Faint cytoplasmic C3aR staining was observed in hepatocytes of WT rats (Figure 5, Table 1). This was substantially augmented in hepatocytes of *Hmox1*−*/*<sup>−</sup> rats. In lung tissue sections, cytoplasmic C3aR staining was observed in bronchial epithelium of WT and *Hmox1*−*/*<sup>−</sup> rats, with a significant reduction of expression in the latter. In spleen tissue, C3aR staining was detected in red and white pulp of both WT and *Hmox1*−*/*<sup>−</sup> rats, with weaker staining in *Hmox1*−*/*<sup>−</sup> rats (Figure 5, Table 1).

**Figure 5.** C3aR staining of WT and *Hmox1*−*/*<sup>−</sup> rat tissue sections. Staining of C3b was detected in all tissue sections examined. Magnification ×200.

#### *3.6. Effect of HO-1 Depletion on Tissue Morphology*

A mild mesangial hypercellularity in kidney tissue was noticed in *Hmox1*−*/*<sup>−</sup> rats (Figure 2). Lung tissue displayed focal inflammation, mainly with peribronchial distribution (Figure 3). Moreover, absence of HO-1 in the liver resulted in periportal inflammatory infiltrates (Figure 2) and "bridging" consisting of inflammatory cells. Specifically, *Hmox1*−*/*<sup>−</sup> rats showed increased portal inflammation consisting mainly of lymphocytes in the liver.

#### **4. Discussion**

In the current study, we used a previously generated and phenotypically characterized HO-1 deficient rat model to identify potential interactions of HO-1 with CRPs and unravel additional HO-1 complement-related functions. In the transgenic model used, complete deletion of *Hmox1* locus via zinc finger nuclease (ZFN)-mediated gene disruption was achieved. Efficiency of *Hmox1* ablation was validated by both western blotting and Realtime PCR amplification, revealing the complete absence of HO-1 protein and mRNA in renal and extra-renal (lung, liver, spleen) tissues of *Hmox1*−*/*<sup>−</sup> rats, respectively [23]. Moreover, HO-1 deficiency was associated with a renal dysfunction phenotype, characterized by both albuminuria and increased serum creatinine, which accompanied specific glomerular histological alterations, defined by mild mesangial expansion and segmental scleroses. Electron microscopy study of glomeruli also revealed segmental effacement of podocytes foot processes. The generation of this transgenic model confirmed that HO-1 regulates DAF in glomeruli, with functional consequences in terms of C3b deposition.

In the kidney, constitutive expression of DAF is restricted exclusively in glomerular epithelial cells (podocytes) [22]. This was verified in the present study, which demonstrated a reduction in DAF staining in glomeruli of *Hmox1*−*/*<sup>−</sup> rats (Figure 1). Our previous work has also demonstrated that HO-1 ablation in transgenic rats resulted in a reduction of glomerular DAF expression [7], while podocyte-specific HO-1 overexpression increased DAF expression and reduced glomerular C3b deposition following administration of the complement fixing antibody anti-GBM.

The assessment of changes in tissue detection of CRPs other that DAF in WT and *Hmox1*−*/*<sup>−</sup> kidneys demonstrated that the most apparent differences were observed for Crry protein, which restricts complement activation via both alternative and classical pathways. A reduction of Crry was observed in *Hmox1*−*/*<sup>−</sup> kidney, liver and lung tissue. In contrast, CD59, which limits C5b-9-mediated cytotoxicity and specifically collaborates with Crry to control complement activation [24], could not be detected in either WT or *Hmox1*−*/*<sup>−</sup> rat tissues examined. Crry has been reported to functionally resemble MCP, while at the structural level, it shares more similarities with CR1, as it is attached on the cell via a transmembrane domain and does not possess any serine-threonine-rich structures [25]. The reduction of DAF and Crry protein staining observed in kidney tissue was associated with increased deposition of C3b (Figure 4), supporting a functional significance of this regulatory effect of HO-1 on these two CRPs.

Regarding the pathological consequences of HO-1 defective activity in renal parenchyma, previous results suggest that HO-1 absence results in lesions resembling focal segmental glomerular sclerosis (FSGS) in the kidney [23], a finding confirmed in the present study. Moreover, it was recently reported that reduced DAF expression may promote development of FSGS [26]. This was verified in DAF knock out mice and a model of adriamycin (ADR) induced nephropathy. The same study also confirmed reduced levels of DAF staining in human tissue specimens from patients with FSGS, with a simultaneous increase in C3b deposition, suggesting that lack of DAF results in FSGS lesions and that injury is primarily mediated by the C3a–C3aR axis [26]. In the present study, we also observed a significant decrease in DAF expression coupled with increased C3b staining in *Hmox1*−*/*<sup>−</sup> rat kidney sections, albeit C3aR staining was reduced compared to WT. This finding suggests that reduced DAF expression may also contribute to the development of FSGS lesions previously reported in HO-1 deficient rats [23]. Reduced immunostaining of DAF and Crry was also observed in *Hmox1*−*/*<sup>−</sup> liver tissue. However, there was no change in C3b detection, suggesting that the reduced expression of DAF and Crry, consequent to HO-1 depletion, in liver tissue was not sufficient to increase complement deposition and that lesions observed (periportal inflammatory infiltrates and bridging) are not mechanistically linked to reduced DAF or Crry levels.

In our study, it was not possible to assess changes in CD59, as this was not detected in any of the tissues. This is in contrast to previous studies, in which CD59 expression has been verified in kidney and stomach tissue samples [27,28]. The reason for this discrepancy could be related to detection methods used.

An effect of HO-1 ablation on C3aR expression was also observed in kidney, lung and spleen tissue, in which this receptor was reduced, and in liver tissue, in which it was augmented. C3aR is a G-protein-coupled receptor [29], via which C3a modulates immunity and certain cancers [30]. It also promotes podocyte autophagy and apoptosis [31]. Although C3aR function is not related to the function of CRPs, it is also attached on the cell membrane due its receptor function. A differential effect of HO-1 depletion on C3aR expression was identified in the present study, depending on the specific tissue, and it indicates a plausible effect of HO-1 on the C3-C3aR axis, thus extending its role as a contributor to the balanced complement cascade activation.

In a previous study assessing expression of complement factors in rat podocytes, increased C3aR expression was observed in glomeruli in puromycin (PAN)-induced renal injury, and this was accompanied by increased glomerular DAF expression [28]. In the present study, we observed decreased DAF immunostaining accompanied by a decrease in that of C3aR in the absence of injury. The reduction in DAF and Crry expression would be expected to increase C3aR expression, as the C3a-C3aR axis mode of action would be stimulated. However, our results indicate that the reduced DAF and Crry expression observed in *Hmox1*−*/*<sup>−</sup> rats was not sufficient to augment C3aR expression.

A mechanism by which HO-1 can regulate expression of CRPs could involve effects on the transcription factor Sp1, which is activated by the heme degradation product, CO [32]. Constitutive CD59 and DAF expression was shown to depend on Sp1 binding to specific promoter regions [33]. Further, the C3aR gene was shown to also include recognition sequences for Sp1 [34].

#### **5. Conclusions**

The present study provides an immunohistochemical characterization of the effect of HO-1 on CRP expression in various tissues. The study assesses the tissue-specific effect of HO-1 on CRP expression and shows a more prominent effect in the kidney, supporting the important role of HO-1 in mediating immune-mediated kidney injury. The observations further support the role of HO-1 as a regulator of complement activation.

**Author Contributions:** Conceptualization, M.G.D.; methodology, M.G.D. and E.T.; formal analysis, M.G.D., K.P., H.G. and E.A.L.; resources, M.G.D., H.G. and E.A.L.; data curation, M.G.D., E.T., K.P. and E.A.L.; writing—original draft preparation, M.G.D.; writing—review and editing, E.A.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a Merit Review Award # BX004333-02 from the United States (U.S.) Department of Veterans Affairs (Biomedical Laboratory Research and Development Service) to E.A.L.

**Institutional Review Board Statement:** The study was approved by the Project Evaluation Committee of the Agricultural and Veterinary Policy Sector of the Greek Ministry of Agricultural Development and Food. Project approval code: 4201. Date of approval: 2 July 2014.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is contained within the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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