Cobalt Protoporphyrin IX Attenuates Antibody-Mediated, Complement-Dependent Podocyte Injury: Role of Cobalt and Porphyrin Moieties

Inorganics · 2025-02-23

Metalloporphyrins (MPs) that induce heme oxygenase (HO)-1 were shown to attenuate complement-mediated glomerular injury, with cobalt protoporphyrin IX (CoPPIX) being the most effective. To decipher the efficacy between CoPPIX and its constituents (Co, PPIX), we compared the outcomes of treatment with each in a rat model of complement-dependent immune injury of glomerular epithelial cells (podocytes). Outcomes were correlated with HO-1 induction and expression levels of complement C3 and of the complement activation regulators (CARs) cluster of differentiation (CD)55, CD59, and CR1-related gene y protein product (Crry). Podocyte injury was induced in rats following a single injection of the complement-fixing antibody against the podocyte antigen, Fx1A. CoPPIX or its constituents, cobaltous chloride (CoCl2) and protoporphyrin IX (PPIX), were injected prior to and on alternate days thereafter. Urine was assessed for protein excretion and kidney cortex samples were processed for histopathology and assessment of target gene mRNA and protein levels using digital polymerase Chain Reaction (dPCR) and capillary-based Western blot analysis. The anti-Fx1A antibody caused proteinuria and podocyte injury. Treatment with the full CoPPIX chelate reduced proteinuria but treatment with either CoCl2 or PPIX did not. CoPPIX treatment potently induced HO-1 and reduced tissue C3 mRNA and protein levels. It also increased CD55, CD59, and Crry mRNA, with an inconsistent effect on protein levels. The Co moiety was required for HO-1 induction but not for the decrease in C3. This decrease did not significantly correlate with the effects of CoPPIX treatment on CD55 protein levels. Chelation of cobalt to PPIX enhanced its potency to induce HO-1 but reduced that on CD55 induction. These observations distinguish between the effects of CoPPIX and its constituents on proteinuria consequent to complement-mediated podocyte injury and underlying mediators and identify this MP as a potential disease-modifying agent.

Metalloporphyrins as Tools for Deciphering the Role of Heme Oxygenase in Renal Immune Injury

International Journal of Molecular Sciences · 2023-04-06 · 4 citations

Renal immune injury is a frequent cause of end-stage renal disease, and, despite the progress made in understanding underlying pathogenetic mechanisms, current treatments to preserve renal function continue to be based mainly on systemic immunosuppression. Small molecules, naturally occurring biologic agents, show considerable promise in acting as disease modifiers and may provide novel therapeutic leads. Certain naturally occurring or synthetic Metalloporphyrins (Mps) can act as disease modifiers by increasing heme oxygenase (HO) enzymatic activity and/or synthesis of the inducible HO isoform (HO-1). Depending on the metal moiety of the Mp employed, these effects may occur in tandem or can be discordant (increased HO-1 synthesis but inhibition of enzyme activity). This review discusses effects of Mps, with varying redox-active transitional metals and cyclic porphyrin cores, on mechanisms underlying pathogenesis and outcomes of renal immune injury.

Immune-Related Functions of Heme Oxygenase-1

Antioxidants · 2023-06-22 · 6 citations

Heme oxygenase (HO)-1 is a well-known cytoprotective enzyme due to its enzymatic action, which involves the catalysis of heme into anti-apoptotic and antioxidant molecules such as bilirubin, biliverdin and CO [...].

Metalloporphyrins Reduce Proteinuria in Podocyte Immune Injury: The Role of Metal and Porphyrin Moieties

International Journal of Molecular Sciences · 2023-08-14 · 5 citations

Depending on their central metal atom, metalloporphyrins (MPs) can attenuate or exacerbate the severity of immune-mediated kidney injury, and this has been attributed to the induction or inhibition of heme oxygenase (HO) activity, particularly the inducible isoform (HO-1) of this enzyme. The role of central metal or porphyrin moieties in determining the efficacy of MPs to attenuate injury, as well as mechanisms underlying this effect, have not been assessed. Using an antibody-mediated complement-dependent model of injury directed against rat visceral glomerular epithelial cells (podocytes) and two MPs (FePPIX, CoPPIX) that induce both HO-1 expression and HO enzymatic activity in vivo but differ in their chelated metal, we assessed their efficacy in reducing albuminuria. Podocyte injury was induced using rabbit immune serum raised against the rat podocyte antigen, Fx1A, and containing an anti-Fx1A antibody that activates complement at sites of binding. FePPIX or CoPPIX were injected intraperitoneally (5 mg/kg) 24 h before administration of the anti-Fx1A serum and on days 1, 3, 6, and 10 thereafter. Upon completion of urine collection on day 14, the kidney cortex was obtained for histopathology and isolation of glomeruli, from which total protein extracts were obtained. Target proteins were analyzed by capillary-based separation and immunodetection (Western blot analysis). Both MPs had comparable efficacy in reducing albuminuria in males, but the efficacy of CoPPIX was superior in female rats. The metal-free protoporphyrin, PPIX, had minimal or no effect on urine albumin excretion. CoPPIX was also the most potent MP in inducing glomerular HO-1, reducing complement deposition, and preserving the expression of the complement regulatory protein (CRP) CD55 but not that of CD59, the expression of which was reduced by both MPs. These observations demonstrate that the metal moiety of HO-1-inducing MPs plays an important role in reducing proteinuria via mechanisms involving reduced complement deposition and independently of an effect on CRPs.

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

Biomedicines · 2023-12-13 · 1 citations

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.

Effect of Heme Oxygenase-1 Depletion on Complement Regulatory Proteins Expression in the Rat

Antioxidants · 2022-12-27 · 2 citations

Heme oxygenase has been implicated in the regulation of various immune responses including complement activation. Using a transgenic rat model of HO-1 depletion, the present study assessed the effect of HO-1 absence on the expression of complement regulatory proteins: decay accelerating factor (DAF), CR1-related gene/protein Y (Crry) and CD59, which act to attenuate complement activation. Protein expression was assessed by immunohistochemistry in kidney, liver, lung and spleen tissues. DAF protein was reduced in all tissues retrieved from rats lacking HO-1 (Hmox1−/−) apart from spleen tissue sections. Crry protein was also reduced, but only in Hmox1−/− kidney and liver tissue. C3b staining was augmented in the kidney and spleen from Hmox1−/− rats, suggesting that the decrease of DAF and Crry was sufficient to increase C3b deposition. The observations support an important role of HO-1 as a regulator of the complement system.

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli

Current Issues in Molecular Biology · 2021-09-07 · 2 citations

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX−) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX− sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

Morphometric Effects of HO-1 Deficiency and Overexpression in Rat Glomeruli and Podocytes

2021-01-01 · 1 citations

Of the various cells comprising the glomerulus (endothelial, mesangial, resident macrophages, and visceral glomerular epithelial cells also known as podocytes), the latter are most vulnerable to various forms of glomerular injury (Diabetes, Hypertension, immune-mediated, etc.) that frequently progress to end-stage kidney disease (ESKD). This is due to their non-replicative, terminally differentiated highly specialized nature. Specifically, they exhibit a high rate of vesicular traffic as evidenced by multiple coated vesicles and coated pits along with their basolateral domain and have a high capacity for protein synthesis and posttranslational modifications because of a well-developed endoplasmic reticulum and a large Golgi apparatus Podocyte depletion consequent to injury is a well-established mechanism underlying glomerular scarring (sclerosis) while progressive glomerulosclerosis leads to ESKD It is, therefore, not surprising that strategies assessing ways to protect podocytes against injury are being explored.

Morphometric Effects of HO-1 Deficiency and Overexpression in Rat Glomeruli and Podocytes.

PubMed · 2021-06-28

Generation of a novel decay accelerating factor (DAF) knock-out rat model using clustered regularly-interspaced short palindromic repeats, (CRISPR)/associated protein 9 (Cas9), genome editing

Transgenic Research · 2021-01-02 · 5 citations