**1. Introduction**

Heme oxygenase (HO)-1 is a well-known cytoprotective enzyme, mainly due to its ability to degrade heme into the established antioxidant and anti-apoptotic metabolites products bilirubin, biliverdin and carbon monoxide (CO). Furthermore, it has been implicated in the regulation of various cellular, biological and immunological responses. As an integral part of cell defense mechanisms against oxidative stress, HO-1 expression is induced in various tissues upon specific stimuli, including hypoxia, inflammatory cytokines or hemolysis, and is regulated by the transcription factor Nrf2 (Nuclear factor erythroid 2-related factor 2). HO-1 is also induced in many pathological conditions, while its expression levels vary greatly between different organs, as well as between the various organ cell types. In the lung, HO-1 induction is detected predominantly in pneumocytes II and in alveolar macrophages in both inflammatory, such as acute respiratory distress syndrome (ARDS), and vascular abnormalities [1], while in the liver it is found primarily in Kupffer cells in conditions of ischemia or toxic injury [2,3]. In the kidneys, it is primarily induced in tubules, both in glomerular and tubulointerstitial diseases of various etiologies [4].

Recently, it was demonstrated that HO-1 regulates complement activation, both by generation of heme degradation products (bilirubin and CO) [5] and by limiting levels of unbound heme, which activates the complement system [6]. This finding implies that HO-1 may possess an important role in limiting excessive complement activation. The complement system is a key mechanism of the innate immunity. Its activation involves mainly three pathways: (i) the classical pathway, in which antibodies bound to antigen lead to the recruitment of the C1 complex; (ii) the alternative pathway, which remains continuously active at a low level due to spontaneous hydrolysis of C3 to C3 (H2O); and (iii) the mannose-binding lectin (MBL) pathway, in which MBL protein binds to its target (for example, mannose on the surface of a bacterium) and MASP (Mannan-binding lectin

**Citation:** Detsika, M.G.; Theochari, E.; Palamaris, K.; Gakiopoulou, H.; Lianos, E.A. Effect of Heme Oxygenase-1 Depletion on Complement Regulatory Proteins Expression in the Rat. *Antioxidants* **2023**, *12*, 61. https://doi.org/ 10.3390/antiox12010061

Academic Editor: Stanley Omaye

Received: 15 November 2022 Revised: 14 December 2022 Accepted: 22 December 2022 Published: 27 December 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

serine protease) 1 cleaves C4 into C4a and C4b. Activation of any of the three pathways leads to the C3 step, at which C3 cleavage by the C3 convertases generates active C3b. Binding of the C3b-active fragment to the C3 convertase results in deactivation of the C3 convertase and formation and activation of the C5 convertase, which cleaves circulating C5 into active C5a and C5b fragments. C5b generation enables activation of the terminal step, in which the membrane attack complex (MAC, C5b-9), a pore-like structure causing lysis of targeted cells, is formed [7–9]. Moreover, C3a and C5a, known to act as anaphylatoxins, recruit and activate various immune cells.

Complement activation is a central mechanism in many renal immune-mediated conditions. Its important role has been demonstrated in experimental models of immune- mediated forms of glomerular injury, such as Heyman nephritis [10], resembling membranous nephropathy, and the anti-glomerular basement membrane (GBM) model [11], resembling anti-GBM disease, also known as Goodpasture's syndrome. It also plays a key role in mediating kidney injury and progression to end-stage renal disease in a plethora of human kidney diseases. C3 glomerulopathy, a recently characterized disease entity that includes dense deposit disease (DDD) and C3 glomerulonephritis (C3 GN), is driven by uncontrolled activation of the alternative complement pathway [12]. Apart from the established role of complement activation in renal diseases, a potential role for complement has also been reported in lung and injury. Increased levels of complement factors have been measured in various conditions leading to lung injury, such as ARDS, pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF) [13]. Complement activation has also been implicated in non-alcoholic fatty acid liver disease (NAFLD) leading to non-alcoholic steatohepatitis (NASH), with increased deposition of complement factors around steatotic hepatocytes and reported to be associated with disease progression and a proinflammatory profile [14]. Finally, a previous study utilizing complement factor H (CFH)-deficient mice reported the development of splenomegaly with distorted spleen architecture [15].

Activation of the complement system is controlled by complement regulatory proteins (CRPs), which ensure maintenance of balanced activation of all three complement pathways by acting at multiple steps of the cascade [16]. Given the fact that activation of C3 is the key step in complement activation, it is not surprising that several of the regulatory proteins act at the C3 convertase step. In the rat, cell membrane-bound CRPs include decay accelerating factor (DAF), CD59 and CR1-related gene/protein Y (Crry) [17], which is considered as the murine analogue of the human membrane cofactor protein (MCP) [18,19]. CRPs are an important group of cell surface glycoproteins and share similar structural and functional characteristics. CRPs are all transmembrane proteins attached on the cell surface, allowing for the remaining part of the protein structure to perform its function. Specifically, DAF and CD59 are attached on the cell membrane via a glycosylphosphatidylinositol (gpi) anchor, while Crry is attached via a transmembrane domain [13]. They are all initially synthesized in the cell nucleus and subsequently undergo heavy N- and O- glycosylation until they finally reach the cell membrane [14,15]. Soluble forms of DAF and CD59 have also been identified, both in humans and rats [7]. Upon anchoring on the cell membrane, DAF and Crry promote the decay of C3 and C5 convertases, thereby limiting the extent of complement cascade activation, whilst CD59 acts solely on the final step of C5b-9 (membrane attack com-plex MAC) formation and activation, thereby limiting cell lysis. Therefore, the balanced activation of the complement cascade in healthy systems largely depends on the functional integrity of these CRPs.

We previously reported a reduction in glomerular DAF expression in HO-1 knock out (*Hmox1*−/−) rats, which was sufficient to minimize C3b deposition upon injury caused by administration of a complement fixing antibody against the GBM [20]. However, the effect of HO-1 depletion on other complement regulatory proteins, both in renal and extra-renal tissues, was not assessed. The present study examined protein levels of CRP (DAF, Crry, CD59), levels of the cleavage product of complement component C3 (C3b), and levels of the complement component 3a receptor (C3aR) in the kidney, lung, liver and spleen of *Hmox1*−/<sup>−</sup> rats.

#### **2. Materials and Methods**

#### *2.1. Animals*

Sprague Dawley rats were reared in accordance to the European Union Directive for care and use of laboratory animals, and all procedures were approved by the Hellenic Veterinary Administration and the ethical committee of 'Evangelismos' Hospital. For generation of transgenic rats, all procedures described were conducted according to the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals.

HO-1 knock out animals (homozygotes, *Hmox1*−/−) were obtained through breeding of *Hmox1*+/<sup>−</sup> rats, generated as previously described [21]. All invasive experimental procedures were carried out under ketamine:xylazine (1: 3, 0.1 mL/100 g body weight) anesthesia.

#### *2.2. Tissue Retrieval and Immunohistochemistry*

Wild type (WT) or *Hmox1*−/<sup>−</sup> kidney, lung, liver and spleen tissue samples were fixed in formalin (30%)-buffered saline for 24 h. Tissues were dehydrated with graded ethanol solutions (80–90%) and embedded in paraffin prior to sectioning (4-μm-thick sections). Sections were applied to Poly-L-Lysine-coated slides and left at 55 ◦C for paraffin excess removal. Sections were incubated with either of anti-rat DAF (clone: RDIII-7), Crry (clone: TLD-1C11), CD59 (clone:TH9), C3/C3b (clone: 2B10B9B2) (Hycult, Uden, Netherlands) and C3aR (clone: D-12), (DTX, Santa Cruz, CA, USA) primary antibodies, followed by incubation with speciesspecific secondary antibodies, using established techniques. Sections incubated in the absence of primary antibody served as negative controls. For the staining process, deparaffinization, rehydration and antigen retrieval were performed by heating the slides in a PT module (PT Link, DAKO, CA, USA) for 20 min at 96 ◦C, at high pH (EnVision FLEX Target Retrieval Solution, High pH (50X), DAKO, Santa Clara, CA, USA). Endogenous peroxidase was blocked by incubating the slides with hydrogen peroxide 3% for 15 min at room temperature. Sections were incubated with the primary antibodies overnight at 4 ◦C, at a dilution of 1:50 for each marker. After rinsing with tris buffer solution, slides were incubated with EnVision FLEX HRP (DAKO, Santa Clara, CA, USA), for 30 min at room temperature. DAB was used as chromogen and was provided by EnVision FLEX KIT (DAKO, Santa Clara, CA, USA). Sections were incubated with DAB for 6 min at room temperature.

#### **3. Results**

## *3.1. Effect of HO-1 Depletion on DAF Expression*

Immunohistochemistry for assessment of DAF expression in *Hmox1*−/<sup>−</sup> tissues revealed reduced DAF staining in kidney, liver, lung and spleen tissue. DAF staining did not differ in *Hmox1*−/<sup>−</sup> spleen tissue compared to WT (Figure 1) (Table 1). Staining for DAF in kidney sections was observed in glomerular epithelial cells (podocytes). This is in line with previous reports demonstrating constitutive expression of DAF exclusively in renal podocytes [22]. DAF staining in the liver was observed in hepatocytes (Figure 1) with a reduction of expression in liver tissue from *Hmox1*−/<sup>−</sup> rats. A reduction of DAF staining was also observed in the lung, in which DAF was mainly detected in the lung parenchyma.

**Table 1.** CRP and C3aR expression in WT and *Hmox1*-/- tissue samples and C3b deposition.


Scale: −, +, ++ indicate no staining, variable staining and marked staining, respectively. ↓ reduced protein expression, ↑ increased protein expression.

**Figure 1.** Immunostaining of DAF in tissue sections obtained from WT and *Hmox1*−/<sup>−</sup> rats. Reduced DAF staining was observed in kidney, liver and lung tissue. No apparent differences in DAF staining in spleen tissue sections were shown in *Hmox1*−/<sup>−</sup> rats. Magnification <sup>×</sup>200.
