ACE2 Deficiency Protects Against Heme Protein-Induced Acute Kidney Injury

Abstract

Angiotensin-converting enzyme 2 (ACE2) exerts countervailing effects on the renin-angiotensin-aldosterone system. ACE2 also engages the spike protein of SARS-CoV-2. ACE2 protein has been shown recently to avidly bind heme. We examined the pathobiologic relevance of this heme-binding property of ACE2 by employing the glycerol-induced model of heme protein-mediated AKI (HP-AKI) which is characterized by increased kidney heme content. We studied the response of ACE2-wildtype (ACE2^+/y) and ACE2-deficient (ACE2^-/y) mice to HP-AKI and quantitated kidney and cellular content of heme under relevant conditions. ACE2-deficient mice, compared with ACE2-wildtype mice, were significantly protected against HP-AKI as reflected by filtration markers, less histologic injury, and less expression of apoptosis and ferroptosis markers. ACE2-deficient mice also evinced lesser kidney heme content and a blunted induction of HO-1. HEK293 ACE2-overexpressing cells, compared with HEK293-native cells, when exposed to heme, retained higher amounts of heme. In HP-AKI, ACE2 expression and activity were reduced, and myoglobin and heme, administered independently, reduced ACE2 expression in the otherwise intact mouse kidney. Finally, with more severe HP-AKI, the protective effect of ACE2 deficiency was attenuated. We conclude that ACE2 deficiency confers protection against HP-AKI. We suggest that this reflects the recently recognized binding of heme to ACE2, such binding serving to facilitate renal entry of heme, a known nephrotoxin. These findings uncover a novel pathway of heme-dependent acute kidney injury. This is the first demonstration of the biologic relevance of chemical binding of heme by ACE2. Finally, we identify heme proteins and heme as novel determinants of ACE2 expression.

Graphical Abstract

Introduction:

At least four considerations underlie the interest in and the importance of angiotensin converting enzyme 2 (ACE2). First, ACE2 exerts countervailing effects when the renin-angiotensin-aldosterone system (RAAS) is activated. ACE2 catalyzes the conversion of angiotensin II to angiotensin(1–7), and in so doing, reduces the levels of angiotensin II and its attendant effects that may be prooxidant, vasoconstrictive, proinflammatory, profibrogenic, and salt-retaining (through its product, aldosterone); additionally, ACE2 promotes the generation of angiotensin(1–7) which exerts vasorelaxant and anti-inflammatory actions (1). Second, ACE2 is the cell surface receptor that engages the spike protein of SARS-CoV-2 and enables the intracellular uptake and subsequent intracellular proliferation of SARS-CoV-2 (1). Third, largely through studies involving ACE2-deficient mice, ACE2 is recognized as protective in models of ischemic acute kidney injury (2), Alport syndrome (3), diabetic kidney disease (4), obesity-related glomerulopathy (5), and angiotensin II-induced nephropathy (6). Fourth, the administration of soluble ACE2 is currently being pursued as a translational strategy in the protection of human kidney disease (3, 7).

During the recent COVID-19 pandemic, several observations were made that, in aggregate, engendered the present study: plasma levels of myoglobin, a heme-containing nephrotoxin, were identified as a significant risk factor for organ failure and mortality in COVID-19 (8); plasma levels of heme are increased in patients with COVID-19, especially those who exhibit desaturation (9); and such elevated plasma levels of heme observed in patients with COVID-19 may attain levels comparable to those observed in patients with hemolytic conditions (10), the latter commonly provoking AKI.

We thus examined the functional significance of ACE2 in a long-established model of heme protein-induced AKI (HP-AKI), hypothesizing that the deficiency of ACE2, as exists in ACE2-deficient (ACE2^-/y) mice, would exacerbate HP-AKI. We used the widely employed model of HP-AKI as induced by the intramuscular injection of hypertonic glycerol. The latter induces rhabdomyolysis, intravascular hemolysis, and AKI because of exposure of the kidney to myoglobin, hemoglobin, and, ultimately, heme. This model, for example, has been previously used to demonstrate the role of pre- and post-glomerular vasoconstriction in AKI (11); induction of heme oxygenase-1 (HO-1) as a protective response in AKI (12); early mitochondrial injury, autophagy, and mitophagy in AKI (13); and the significance and potency of ferritin as a protectant against AKI (14). Using this HP-AKI model and based on the recognized nephroprotective effects of ACE2 in assorted models of kidney injury, we hypothesized that ACE2-deficient (ACE2^-/y) mice, compared with ACE2-wildtype (ACE2^+/y) mice, would exhibit increased sensitivity to HP-AKI.

Materials and Methods:

In Vivo studies:

All studies were approved by the Institutional Animal Care and Use Committee of Mayo Clinic and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. We utilized male Sprague Dawley rats (250–275g) purchased from Inotiv (Indianapolis, IN) and male C57BL6/J mice (10–15 wk old) purchased from Jackson Lab (Bar Harbor, ME) for these studies employing the glycerol model of HP-AKI. Additionally, male ACE2-deficient mice (ACE2^-/y, 10–20 wk old), with targeted deletion of ACE2, an X chromosome-linked gene, as previously described by Gurley et al (15) and contemporaneous wildtype littermate controls (ACE2^+/y) were similarly employed. In all studies, groups of rats or mice were age- and/or weight-matched for random assignment to control or experimental groups, accordingly.

HP-AKI Model:

Rats and mice were subjected to the glycerol model of heme protein-mediated acute kidney injury (HP-AKI) as we described previously (12,16). Briefly, after overnight dehydration (~18 hours), mice or rats were given an intramuscular injection of glycerol (50% in water, 6 ml/kg for rats, and 6 or 5 ml/kg for mice) under ketamine and xylazine, anesthesia (IP, 90 and 10 mg/kg in mice and 50 and 5 mg/kg in rats, for ketamine and xylazine, respectively). At 1 or 2 days after glycerol injection, rats and mice were euthanized and kidney tissue and plasma were collected for analysis as outlined below. Kidney function was assessed by the measurement of plasma creatinine and BUN levels as previously described (16).

Administration of Heme Protein or Hemin:

Intravenous administration of myoglobin (250 mg/100 g body weight, catalog no. M0630, Sigma Aldrich, St. Louis, MO) was performed in C57BL/6J mice, after 16–18 hours of dehydration. In additional studies, hemin (hemin ferriprotoporphyrin IX chloride, 50 μmol/kg, IP) was administered to C57BL/6J mice at 6 and 24h before kidneys were harvested for gene expression studies (16).

Histological Studies:

Histologic examination was performed on formalin-fixed, paraffin-embedded kidney sections stained with hematoxylin and eosin (16). Histologic alterations were assessed by semiquantitative scoring of necrosis, cast formation, tubule dilation, and overall corticomedullary injury.

Immunofluorescence Staining:

Immunofluorescence staining was performed on 8 μm sections cut from formalin-fixed, paraffin-embedded kidney tissues as described (17). Briefly, slides were deparaffinized and antigen retrieval with acidic citrate buffer (pH 6.0) was performed. After quenching in 100 mM NH₄Cl for 20 minutes and blocking (5% normal donkey serum, 5% BSA in 0.1% Triton X-100, PBS) for 2 hours, slides were incubated with primary antibodies at 4°C overnight. The next day the secondary antibody incubation was performed at room temperature for 2 hours. Washes using PBS + 0.05% Triton X were performed between the incubations. Primary antibodies against ACE2 (catalog NBPI-76614, Novus Biologicals, Centennial, CO) and KIM1 (catalog AF1817, R&D Systems, Minneapolis, MN) and secondary antibodies (catalog nos. A32814 and A32794, ThermoFisher Scientific) were used. Microscope images (Zeiss LSM980) were acquired using a 20X lens (NA 0.3) and images were prepared using Photoshop. All exposure levels were identical among the groups. Supplemental Table 1 provides RRID information for antibodies used in these studies.

Gene and protein expression:

Kidney mRNA and protein expression was assessed as previously described (16). Briefly, a two-step real-time RT-PCR method employing a Transcriptor First Strand cDNA synthesis kit (Roche, Indianapolis, IN) and TaqMan Gene Expression assays (ThermoFisher Scientific) was used for mRNA quantitation. Protein expression for ACE2, BCL2, and cPARP was assessed by Western blot analysis using overnight 4°C incubations of primary antibodies. Primary antibodies employed targeted ACE2 (catalog no. NBP1–76614, Novus Biologicals), BCL2 (catalog no. 28150, Cell Signaling), and cPARP (catalog no. 9542, Cell Signaling Technology, Danvers, MA). Normalization for densitometric analysis of Western blots was performed by assessment of total protein staining (mouse) or GAPDH (catalog no. 2118, Cell Signalling) expression (rat). Supplemental Table 1 provides information for TaqMan Gene Expression assays and antibodies used in these studies.

ACE2 activity:

As described in detail in our previous study, renal ACE2 activity was quantified using a fluorometric method (17). Briefly, tissues were homogenized (1:10 w/v) in lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 25 μM ZnCl₂, and 1mM PMSF) and supernatants were prepared by centrifugation (10,000 g, 4°C). Supernatants (2 mg protein) were assayed in reactions containing: 50 mM 4-Morpholineethanesulfonic acid, pH 6.5, 300 mM NaCl, 10 μM ZnCl₂, 20 μM captopril, 0.01% Triton X, and 30 μM substrate (Mca-APK[Dnp], Enzo Life Sciences, Farmingdale, NY) and an EDTA-free protease inhibitor tablet (catalog 11873580001, Roche Diagnostics). Fluorescence generation was assessed over 40 minutes (FluoroSkan plate reader, ThermoFisher Scientific, Ex/Em:320/415) for each sample in the presence and absence of the ACE2 inhibitor MLN-4760 (250 μM, catalog 5.30616.0001, EMD Millipore, Burlington, MA). ACE2-specific activity (Δ fluorescence generated: absence minus presence of ACE2 inhibitor) was calculated and expressed as RFU/min/μg protein.

Heme Content:

Heme content in kidney tissue and cell lysates was quantified as in our previous study (13) using the pyridine hemochromogen method. Briefly, tissue and cells were homogenized in 0.1 M potassium phosphate (pH7.4), and lysates were prepared by centrifugation at 500 x g for 5 min at 4°C. Reaction mixtures consisting of 1 mg (tissues) or 0.5 mg (cells) lysate protein, 18% pyridine, and 0.09 N sodium hydroxide were prepared in duplicate, and either sodium dithionite (2 to 3 mg, reducing agent) or potassium ferricyanide (3 μL, 0.1 M, oxidizing agent) was added. The absorbances of the reduced samples were scanned between 570 and 520 nm wavelength using the oxidized samples as blanks. The difference between the maximum and the minimum (557 and 541 nm, respectively) was determined, and, using the extinction coefficient of 20.7 ml/μmol cm, heme concentration was calculated and standardized per mg protein (BCA assay).

In vitro studies:

Native HEK293 (HEK293-native) cells and HEK293-ACE2 overexpressing (ACE2⁺ HEK293) cells generated by Dr. Michael A. Barry’s laboratory, were maintained as described (18). Briefly, these cell lines were maintained in DMEM containing 10% FBS with the selection antibiotic G418 (0.5 mg/ml) added to maintain HEK293-ACE2⁺ cells. For these studies, cells were incubated with DMEM (no phenol red) + 0.1% FBS with hemin (2 μM, Frontier Specialty Chemicals, Logan, UT) for 4 hours after which they were lysed in potassium phosphate buffer (0.1 M, pH 7.4). Supernatants prepared by centrifugation (500 x g, 5 min) were assayed for heme content as described above.

Statistics:

Data are expressed as mean ± SEM and considered statistically significant for P<0.05. The Student’s t-test was used for parametric data and the Mann–Whitney U-test was used for nonparametric data.

Results

Our initial studies employed glycerol-induced HP-AKI at a dose of 6 ml/kg. At this dose, we observed that, very much to our surprise, ACE2-deficient mice, compared with ACE2-wildtype mice, exhibited a significantly lower serum creatinine (2.19±0.12 vs 2.65±0.06 mg/dL, P=0.0096); BUN, while numerically lower, was not significantly altered in these two groups (148.8±5.61 vs 155.9±6.88 mg/dL).

We considered the possibility that, if indeed the deficiency of ACE2 exerted a protective effect in this model, then a lower dose of glycerol that still caused HP-AKI but not overwhelmingly so, may unmask any such protection that is thereby conferred by the deficiency of ACE2. We thus used glycerol-induced HP-AKI at a dose of 5 ml/kg. At this dose, while serum creatinine and BUN were elevated in both groups in HP-AKI, ACE2-deficient mice, compared with ACE2-wildtype mice, exhibited significantly lower serum creatinine and BUN (Figure 1A). Along with less functional impairment, ACE2-deficient mice compared with ACE2-wildtype mice, subjected to HP-AKI, exhibited less renal histologic injury as reflected by lower scores for cast formation (0.67±0.21 vs 1.67±0.33, P=0.0196), necrosis (0.67±0.21 vs 1.67±0.21, P=0.0183), dilatation (0.83±0.31 vs 2.67±0.21, P=0.0063), and histologic injury at the corticomedullary junction (1.00±0.37 vs 2.83±0.31, P=0.0033), the latter representing a kidney zone where AKI often begins and is most prominent (n=6 in each group). These differences are displayed in representative histologic sections of the kidney cortex and corticomedullary junction in ACE2-wildtype mice (Figure 1B, i and iii, respectively) and in ACE2-deficient mice (Figure 1B, ii and iv, respectively) subjected to HP-AKI.

Figure 1. Kidney function and histology in ACE2-wildtype (ACE2^+/y) and ACE2-deficient (ACE2^-/y) mice subjected to HP-AKI.

A. Serum creatinine (left), and BUN (right) concentration at day 1 after HP-AKI or sham. n=6 in each group; **P<0.01 and ***P< 0.005 vs ACE2^+/y HP-AKI. B. Representative renal histologic sections of the cortex (i) and corticomedullary junction (iii) in ACE2^+/y mice and of the cortex (ii) and corticomedullary junction (iv) of ACE2^-/y mice, at day 1 following HP-AKI. Scale Bar = 100μm.

We then examined markers for apoptosis (Bax/Bcl2 ratio) and ferroptosis (PTGS2 and CHAC1). While these markers were increased in HP-AKI, they were significantly lower in ACE2-deficient mice compared with ACE2-wildtype mice subjected to HP-AKI (Figure 2A, B, and C). We also provide congruent evidence based on protein expression of cell death-related proteins. First, in ACE2^+/y mice subjected to HP-AKI, BCL2, which blocks cell death, is decreased by more than 50% compared with sham-treated ACE2^+/y mice, whereas such BCL2 expression in ACE2^-/y subjected to HP-AKI is essentially unchanged from baseline expression in sham-treated ACE2^-/y mice and is significantly greater than that in ACE2^+/y mice subjected to HP-AKI (Figure 2D). Second, in response to HP-AKI, the cell death marker cleaved PARP (cPARP) is strongly induced in ACE2^+/y mice but is not induced in ACE2^-/y mice subjected to HP-AKI (Figure 2E).

Figure 2. Apoptosis and ferroptosis gene expression and protein expression of cell death-related proteins in the kidneys of ACE2-wildtype (ACE2^+/y) and ACE2-deficient (ACE2^-/y) mice subjected to HP-AKI.

BAX/BCL2 mRNA expression ratio (A), PTGS2 mRNA expression (B), and CHAC1 mRNA expression (C) in the kidneys of ACE2^+/y and ACE2^-/y mice at day 1 after HP-AKI or sham. n=6 in each group; **P<0.01 and ***P<0.005 vs ACE2^+/y HP-AKI. Western bot analysis of BCL2 (D) and cPARP (E) protein expression in the kidneys of ACE2^+/y and ACE2^-/y mice at day 1 after HP-AKI or sham. Mean normalized densitometric values are displayed below each Western blot and total protein staining employed for these analyses is provided in Supplemental Figure 3. n=6 in each group; *P<0.05, and **P<0.01 vs ACE2^+/y HP-AKI.

To examine a possible basis for this protective effect of ACE2 deficiency, we drew upon the fascinating recent observations that ACE2 potently binds heme (10, 15). In HP-AKI, a significant driver of kidney injury are heme proteins (myoglobin and hemoglobin) in general, and heme, in particular. We considered the possibility that the basis for the protective effect of ACE2 deficiency would be less binding of heme by ACE2 (which resides on the apical surface of and in the cytoplasm of renal epithelial cells), with less entry of heme into the plasma membrane. We thus measured heme content in the kidneys of ACE2-wildtype and ACE2-deficient mice subjected to glycerol-induced HP-AKI. As demonstrated in Figure 3A, heme content was increased in HP-AKI, but significantly less so in ACE2-deficient mice compared with ACE2-wildtype mice.

Figure 3. Assessment of heme and HO-1 mRNA in the kidney of ACE2-wildtype (ACE2^+/y) and ACE2-deficient (ACE2^-/y) mice subjected to the HP-AKI and in ACE2-overexpressing cells exposed to heme.

Kidney heme content (A) and HO-1 mRNA expression (B) in the kidneys of ACE2^+/y and ACE2^-/y mice at day 1after HP-AKI or sham. n=6 in each group; *P< 0.05 and ***P<0.005 vs ACE2^+/y HP-AKI. C. Cellular heme content in HEK293-native and HEK293-ACE2⁺ cells following 4-hour hemin exposure. n=6 and n=8 in vehicle-treated and hemin-treated groups, respectively. ****P<0.0001 vs hemin-treated HEK293-native.

In HP-AKI, heme oxygenase-1 (HO-1) is markedly induced, in large part because of the increased kidney content of heme; induction of HO-1 may be used as a readout of heme content in HP-AKI. As demonstrated in Figure 3B, the induction of HO-1 was significantly blunted in the kidneys of ACE2-deficient mice compared with ACE2-wildtype mice subjected to HP-AKI.

To examine further the capacity of ACE2 to bind heme, we employed HEK293-native and genetically altered HEK293-ACE2⁺ overexpressing cells which were used in our prior studies (18). We exposed these cells to heme (2 μM), after which heme content within these cells was measured. We reasoned that overexpression of ACE2 would exaggerate cellular heme binding and heme content in these cells. As shown in Figure 3C, heme content in HEK293-ACE2⁺ cells was significantly higher than in HEK293-native cells.

Finally, we asked whether the expression of ACE2 is altered in glycerol-induced HP-AKI. ACE2 mRNA (Figure 4A), protein (Supplemental Figure 1) and activity (Figure 4B) were all decreased in HP-AKI. Immunofluorescence studies demonstrated cellular and apical expression of ACE2 protein in the sham kidney (Figure 4C i), with decreased ACE2 expression in HP-AKI (Figure 4C iii), along with increased expression of KIM-1, a marker of kidney injury (Figure 4C iv vs ii). To explore the basis for decreased ACE2 expression in HP-AKI, we examined whether heme proteins or heme altered kidney ACE2 expression. Renal ACE2 expression decreased following the administration of myoglobin (Figure 4D), or heme (Figure 4E), compared with the respective vehicle-treated mice.

Figure 4. ACE2 expression in response to HP-AKI and after administration of myoglobin or heme in mice.

A. ACE2 mRNA expression in the kidneys of ACE2^+/y and ACE2^-/y mice at day 1 after HP-AKI or sham. n=6 in each group; ***P<0.005 vs ACE2^+/y sham. B. ACE2 activity in the kidneys of C57BL6/J mice at day 1 after HP-AKI or sham. n=6 and n=7 in sham and HP-AKI groups, respectively; ****P<0.0001 vs sham. C. Immunofluorescence staining for ACE2 (red, i and iii) and KIM1 (green, ii and iv) in C57BL6/J mice at day 1 after to sham (i and ii) or HP-AKI (iii and iv). Scale Bar = 100μm. D and E. ACE2 mRNA expression in the kidneys of C57BL6/J mice at day 1 after administration of myoglobin (D) or hemin (E); n=4 for each of the saline-treated groups and n=4 and n=5 for the myoglobin-treated and hemin-treated groups, respectively. *P<0.05 vs respective Saline.

In glycerol-induced HP-AKI, when induced in the rat, we also demonstrate decreased ACE2 mRNA, protein, and activity (Supplemental Figure 2)

DISCUSSION

In our preliminary studies we noted that the behavior of ACE2-deficient mice was contrary to what we hypothesized: ACE2-deficient mice exhibited a modicum of protection against glycerol-induced HP-AKI when the latter was administered at a relatively high dose. This surprising outcome led us to undertake the present work which, in essence, revealed that ACE2-deficient mice, when subjected to less severe glycerol-induced HP-AKI, and compared with similarly exposed ACE2-wildtype mice, exhibited a clearcut resistance to HP-AKI, as reflected by GFR markers, renal histologic injury, and indices of apoptosis and ferroptosis.

To explore a possible basis for this effect of ACE2 deficiency, we considered recent findings made during and at the end of the COVID-19 pandemic: levels of heme in ill patients with COVID-19 approximate levels observed during hemolytic diseases (10); and SARS-CoV2-related proteins, including ACE2, interact with heme (19). As regards the latter, it was shown using a computational approach that several heme-binding sites were present in the ACE2 protein. These conclusions were supported by studies using UV-Vis spectroscopy. Subsequent studies using surface plasmon resonance spectroscopy and in silico analyses corroborated the binding of heme to heme-binding sites in the ACE2 protein (19).

For several reasons, we were drawn to this observation. In glycerol-induced HP-AKI, kidney content of heme is markedly increased and because of its prooxidant, proinflammatory, and pro-apoptotic effects, such elevation in kidney heme content is a determinant of HP-AKI (20). For example, strategies that diminish the augmentation in heme content by inducing the heme-degrading enzyme, HO-1, reduce HP-AKI, whereas those strategies that further augment heme content by inhibiting HO-1 worsen HP-AKI (12). Heme is elevated in HP-AKI in at least two ways. First, myoglobin and dimeric hemoglobin are readily filtered into the urinary space, and these proteins are incorporated into the kidney via the megalin and cubilin receptors on the apical surface of the proximal tubule; once in the intracellular compartment, these heme proteins release their heme moiety (20). Second, as myoglobin and dimeric hemoglobin appear in the urinary space, they undergo oxidative denaturation that is driven by hydrogen peroxide present in urine, the latter attaining concentrations as high as 100 μM (21). Such oxidative denaturation yields, progressively, metmyoglobin and methemoglobin, hemichromes, and ultimately free heme (20). ACE2 exists in the cytoplasm and apical membrane of the proximal tubular epithelium and is thus exposed to copious quantities of heme. We suggest that in view of the recently described binding of heme to ACE2 (10, 19), heme attaches to ACE2 that resides on the apical domain of proximal tubular epithelial cells. Heme is known to peroxidate membrane lipid and denature membrane-residing proteins, the latter effects destabilizing plasma membrane structure and compromising plasma membrane function (20). Additionally, we speculate that, through the well-recognized intracellular trafficking system, heme, brought into the plasma membrane by ACE2, may gain access to the intracellular compartment (22). ACE2, as it were, inadvertently behaves as a Trojan horse in HP-AKI in affording entry of heme into the kidney. Studies of urinary excretion of heme are of interest as ACE2-mediated renal incorporation of heme would predict increased urinary excretion of heme in ACE2^-/y mice subjected to HP-AKI compared with such excretory rates in ACE2^+/y mice subjected to HP-AKI.

Seminal studies conducted some 50 years ago using this HP-AKI model demonstrated vasoconstriction both in the pre-glomerular and post-glomerular circulations (11). Subsequent literature demonstrated that such vasoconstriction results from the scavenging of vascular nitric oxide by heme proteins; and by oxidant, heme-driven increased synthesis of endothelin, superoxide anion, and isoprostanes, all of which are vasoconstrictive. Such vasoconstricting mechanisms prevail over the vasodilating effects of ACE2 (and other endogenous vasodilators) such that the net characteristic vasoconstriction profile results in this model. This vasoactive imbalance would be further accentuated by the deceased ACE2 activity we now demonstrate in the present studies in this model. Additionally, we speculate that the loss of ACE2 in ACE2^-/y mice interrupts ACE2-mediated heme entry into the kidney and, consequently, oxidant, heme-driven vasoconstrictive mechanisms.

In the glycerol-induced HP-AKI model both in the mouse and the rat, we noted that ACE2 expression and activity were decreased. We identify for the first time novel determinants of ACE2 expression as myoglobin and heme, each independently, suppress renal expression of ACE2 in vivo; as kidney content of myoglobin and heme are both significantly increased in HP-AKI, this may explain the suppression of ACE2 expression we observed in HP-AKI. Studies in vitro and in cells from nonrenal tissue have identified the following as suppressors of ACE2 expression: angiotensin II (23), endothelin-1 (23), ERK1/ERK2 (23), HIF1a (24), and FGF23 (25).

In the clear majority of studies in the literature, ACE2 has been shown to be a protectant in disease models. However, quite uncommonly in the literature, there are descriptions of the deficiency of ACE2 exerting a protective effect in tissue injury (26, 27). Such contrasting functional effects may reflect what is a truism in the field of cytoprotection in the kidney and other tissues, that is, the behavior of a putative protectant is dependent on context and conditions, the type and severity of the imposed insult, and relative properties of the cytoprotectant (28). ACE2 exerts established protective effects by virtue of its vasorelaxant, antioxidant, and anti-inflammatory actions. Offsetting these effects is the capacity of ACE2 to bind heme (10, 19), and as we posit, the capacity of ACE2 to thereby afford heme access to the kidney cells with attendant injurious effects. The functional outcome is dependent upon the interplay of these offsetting effects and their relative dominance. To place these competing effects in the context of our present findings, we offer the speculation that in moderately severe HP-AKI (glycerol-induced HP-AKI of 5 ml/kg), the injurious effect of ACE2 in binding heme (and thereby affording heme access to kidney cells) is dominant over the potentially cytoprotective effects of ACE2. However, as the severity of HP-AKI increases (glycerol-induced HP-AKI of 6 ml/kg), it is conceivable that the heme-binding effect of ACE2 becomes less functionally significant because of either one of two reasons. First, HO-1 induction is dose-dependent in HP-AKI, and it is thus possible that with higher doses of HP-AKI, there is greater induction of HO-1 and more efficient vitiation of the ACE2-heme binding pathway. Second, it is possible that the ACE2-heme-binding is saturable such that with higher doses of HP-AKI, there is no additional ACE2-mediated heme entry into the kidney. In the event of either, the functional effect of ACE2-heme pathway may be overridden by the cytoprotective effects of ACE2. Perhaps other considerations in terms of functional outcome include our novel finding that heme proteins and heme suppress ACE2 expression (thereby attenuating the expression of a putative cytoprotectant), and that heme is known to directly denature proteins (20), the latter raising the possibility that the binding of heme to ACE2 alters the functional properties of ACE2 in general and its cytoprotective properties in particular.

We conclude by highlighting an unexpected and novel setting wherein the expression of ACE2 appears to be, not a protector against, but a clear promoter of, tissue injury - this setting is HP-AKI of moderate severity. The putative mechanism involves the recent novel description of the chemical binding of heme by ACE2, and, as such, the present observations provide the first demonstration that this seminal chemical discovery is relevant to the pathogenesis of disease in general and to heme/heme protein-mediated tissue injury in particular; these findings uncover a previously unrecognized pathway for heme-dependent AKI. In this regard, as ACE2 is expressed by cells other than those in the kidneys, this mechanism of ACE2-heme binding may cause injury in other tissues. Additionally, we identify heme proteins and heme as novel determinants of ACE2 expression. Finally, the present observations may offer an explanation as to why plasma levels of myoglobin and heme in patients with COVID-19 are determinants of poor outcomes in patients infected with SARS-CoV-2.

Supplementary Material

https://doi.org/10.6084/m9.figshare.28611575.v1

News and Newsworthy:

ACE2 protein binds heme, which we reasoned would promote heme entry into the kidney and, accordingly, heme protein-mediated acute kidney injury. Our findings support this hypothesis. This study is the first to demonstrate the biologic relevance of ACE2-heme binding, uncover a new pathway of heme-dependent kidney injury, and identify myoglobin and heme as novel determinants of ACE2 expression. Our study explains why plasma levels of myoglobin and heme predict poor outcomes in patients with COVID-19.