Peroxidasin is required for full viability in development and for maintenance of tissue mechanics in adults.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Mechanism of peroxidasin inactivation in hyperglycemia: Heme damage by reactive oxygen species.

Diabetic complications present a serious health problem. Functional damage to proteins due to post-translational modifications by glycoxidation reactions is a known factor contributing to pathology. Extracellular proteins are especially vulnerable to diabetic damage because robust antioxidant defenses are lacking outside the cell. We investigated glucose-induced inactivation of peroxidasin (PXDN), a heme protein catalyzing sulfilimine crosslinking of collagen IV that reinforce the basement membranes (BM). Experiments using physiological diabetic glucose levels were carried out to exclude several potential mechanisms of PXDN inactivation i.e., direct adduction of glucose, reactive carbonyl damage, steric hindrance, and osmotic stress. Further experiments established that PXDN activity was inhibited via heme degradation by reactive oxygen species. Activity of another extracellular heme protein, myeloperoxidase, was unaffected by glucose because its heme was resistant to glucose-induced oxidative degradation. Our findings point to specific mechanisms which may compromise BM structure and stability in diabetes and suggest potential modes of protection.

Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

Identification of brominated proteins in renal extracellular matrix: Potential interactions with peroxidasin.

Peroxidasin (PXDN) is an extracellular peroxidase, which generates hypobromous acid to form sulfilimine cross-links within collagen IV networks. We have previously demonstrated that mouse and human renal basement membranes (BM) are enriched in bromine due to PXDN-dependent post-translational bromination of protein tyrosine residues. The goal of the present study was identification of specific brominated sites within renal BM. A comprehensive analysis of brominated proteome of mouse glomerular matrix had been performed using liquid chromatography-tandem mass spectrometry. We found that out of over 200 identified proteins, only three were detectably brominated, each containing a single distinct brominated tyrosine site i.e., Tyr-1485 in collagen IV α2 chain, Tyr-292 in TINAGL1 and Tyr-664 in nidogen-2. To explain this highly selective bromination, we proposed that these proteins interact with PXDN within the glomerular matrix. Experiments using purified proteins demonstrated that both TINAGL1 and nidogen-2 can compete with PXDN for binding to collagen IV and that TINAGL1 can directly interact with PXDN. We propose that a protein complex, including PXDN, TINAGL1, nidogen-2 and collagen IV, may exist in renal BM.

Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

Kidney collecting duct cells make vasopressin in response to NaCl-induced hypertonicity.

Vasopressin has traditionally been thought to be produced by the neurohypophyseal system and then released into the circulation where it regulates water homeostasis. The questions of whether vasopressin could be produced outside of the brain and if the kidney could be a source of vasopressin are raised by the syndrome of inappropriate antidiuretic hormone secretion (vasopressin). We found that mouse and human kidneys expressed vasopressin mRNA. Using an antibody that detects preprovasopressin, we found that immunoreactive preprovasopressin protein was found in mouse and human kidneys. Moreover, we found that murine collecting duct cells made biologically active vasopressin, which increased in response to NaCl-mediated hypertonicity, and that water restriction increased the abundance of kidney-derived vasopressin mRNA and protein expression in mouse kidneys. Thus, we provide evidence of biologically active production of kidney-derived vasopressin in kidney tubular epithelial cells.

Peroxidasin Deficiency Re-programs Macrophages Toward Pro-fibrolysis Function and Promotes Collagen Resolution in Liver.

BACKGROUND & AIMS: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix proteins. Extracellular matrix stabilization influences the speed of tissue recovery. Here, we studied the expression and function of peroxidasin (PXDN), a peroxidase that uses hydrogen peroxide to cross-link collagen IV during liver fibrosis progression and regression. METHODS: Mouse models of liver fibrosis and cirrhosis patients were analyzed for the expression of PXDN in liver and serum. Pxdn-/- and Pxdn+/+ mice were either treated with carbon tetrachloride for 6 weeks to generate toxin-induced fibrosis or fed with a choline-deficient L-amino acid-defined high-fat diet for 16 weeks to create nonalcoholic fatty liver disease fibrosis. Liver histology, quantitative real-time polymerase chain reaction, collagen content, flowcytometry and immunostaining of immune cells, RNA-sequencing, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A. RESULTS: In human and mouse cirrhotic tissue, PXDN is expressed by stellate cells and is secreted into fibrotic areas. In patients with nonalcoholic fatty liver disease, serum levels of PXDN increased significantly. In both mouse models of liver fibrosis, PXDN deficiency resulted in elevated monocyte and pro-fibrolysis macrophage recruitment into fibrotic bands and caused decreased accumulation of cross-linked collagens. In Pxdn-/- mice, collagen fibers were loosely organized, an atypical phenotype that is reversible upon macrophage depletion. Elevated ROS in Pxdn-/- livers was observed, which can result in activation of hypoxic signaling cascades and may affect signaling pathways involved in macrophage polarization such as TNF-a via NF-kB. Fibrosis resolution in Pxdn-/- mice was associated with significant decrease in collagen content and improved liver function. CONCLUSION: PXDN deficiency is associated with increased ROS levels and a hypoxic liver microenvironment that can regulate recruitment and programming of pro-resolution macrophages. Our data implicate the importance of the liver microenvironment in macrophage programming during liver fibrosis and suggest a novel pathway that is involved in the resolution of scar tissue.

Peripheral Insulin Resistance Is Associated with Copeptin in Patients with Chronic Kidney Disease.

Background: Insulin resistance is associated with cardiovascular disease risk and worsened kidney function. Patients with CKD have higher levels of insulin resistance. Elevated levels of copeptin (a surrogate for vasopressin levels) have been associated with an increased incidence and progression of CKD, and with incident diabetes mellitus. The purpose of our study was to examine the relationship between insulin resistance, copeptin, and CKD. Methods: We performed a cross-sectional study to investigate if insulin resistance was associated with higher copeptin levels in nondiabetic patients with stage 3-4 CKD versus controls. We measured plasma copeptin levels and used data from 52 patients with stage 3-4 CKD and 85 controls (eGFR ≥60 ml/min per 1.73 m2) enrolled in the Insulin Resistance in Chronic Kidney Disease (IRCKD) study. We then used a multivariable linear-regression model to assess the independent relationship between peripheral or hepatic insulin resistance and copeptin across levels of eGFR. Results: We found that in patients with CKD (eGFR of 30-60 ml/min per 1.73 m2), but not in controls, peripheral insulin resistance was significantly correlated with higher levels of log copeptin (r=-0.21, P=0.04). In patients with CKD, when adjusted for age, sex, BMI, serum osmolality, log IL6, and log leptin/adiponectin ratio, each 1 SD decrease in insulin sensitivity was associated with a 39% increase in serum copeptin levels. The relationship between hepatic insulin resistance, copeptin, and eGFR is similar between controls and patients with reduced eGFR. Conclusion: Peripheral insulin resistance is associated with elevated copeptin levels in nondiabetic patients with stage 3-4 CKD. Further research into how the interaction between peripheral insulin resistance and elevated vasopressin affects CKD progression could be of interest.

The Corrected Serum Sodium Concentration in Hyperglycemic Crises: Computation and Clinical Applications.

In hyperglycemia, hypertonicity results from solute (glucose) gain and loss of water in excess of sodium plus potassium through osmotic diuresis. Patients with stage 5 chronic kidney disease (CKD) and hyperglycemia have minimal or no osmotic diuresis; patients with preserved renal function and diabetic ketoacidosis (DKA) or hyperosmolar hyperglycemic state (HHS) have often large osmotic diuresis. Hypertonicity from glucose gain is reversed with normalization of serum glucose ([Glu]); hypertonicity due to osmotic diuresis requires infusion of hypotonic solutions. Prediction of the serum sodium after [Glu] normalization (the corrected [Na]) estimates the part of hypertonicity caused by osmotic diuresis. Theoretical methods calculating the corrected [Na] and clinical reports allowing its calculation were reviewed. Corrected [Na] was computed separately in reports of DKA, HHS and hyperglycemia in CKD stage 5. The theoretical prediction of [Na] increase by 1.6 mmol/L per 5.6 mmol/L decrease in [Glu] in most clinical settings, except in extreme hyperglycemia or profound hypervolemia, was supported by studies of hyperglycemia in CKD stage 5 treated only with insulin. Mean corrected [Na] was 139.0 mmol/L in 772 hyperglycemic episodes in CKD stage 5 patients. In patients with preserved renal function, mean corrected [Na] was within the eunatremic range (141.1 mmol/L) in 7,812 DKA cases, and in the range of severe hypernatremia (160.8 mmol/L) in 755 cases of HHS. However, in DKA corrected [Na] was in the hypernatremic range in several reports and rose during treatment with adverse neurological consequences in other reports. The corrected [Na], computed as [Na] increase by 1.6 mmol/L per 5.6 mmol/L decrease in [Glu], provides a reasonable estimate of the degree of hypertonicity due to losses of hypotonic fluids through osmotic diuresis at presentation of DKH or HHS and should guide the tonicity of replacement solutions. However, the corrected [Na] may change during treatment because of ongoing fluid losses and should be monitored during treatment.

Peroxidasin-mediated bromine enrichment of basement membranes.

Bromine and peroxidasin (an extracellular peroxidase) are essential for generating sulfilimine cross-links between a methionine and a hydroxylysine within collagen IV, a basement membrane protein. The sulfilimine cross-links increase the structural integrity of basement membranes. The formation of sulfilimine cross-links depends on the ability of peroxidasin to use bromide and hydrogen peroxide substrates to produce hypobromous acid (HOBr). Once a sulfilimine cross-link is created, bromide is released into the extracellular space and becomes available for reutilization. Whether the HOBr generated by peroxidasin is used very selectively for creating sulfilimine cross-links or whether it also causes oxidative damage to bystander molecules (e.g., generating bromotyrosine residues in basement membrane proteins) is unclear. To examine this issue, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to define the distribution of bromine in mammalian tissues. We observed striking enrichment of bromine (79Br, 81Br) in basement membranes of normal human and mouse kidneys. In peroxidasin knockout mice, bromine enrichment of basement membranes of kidneys was reduced by ∼85%. Proteomic studies revealed bromination of tyrosine-1485 in the NC1 domain of α2 collagen IV from kidneys of wild-type mice; the same tyrosine was brominated in collagen IV from human kidney. Bromination of tyrosine-1485 was reduced by >90% in kidneys of peroxidasin knockout mice. Thus, in addition to promoting sulfilimine cross-links in collagen IV, peroxidasin can also brominate a bystander tyrosine. Also, the fact that bromine enrichment is largely confined to basement membranes implies that peroxidasin activity is largely restricted to basement membranes in mammalian tissues.

Quantitating excess tissue sodium.

Using changes in tissue [Na+] concentration alone as done with Na+ MRI may not accurately quantitate excess tissue Na+, particularly in cellular tissues. However, individually quantitating alterations in tissue Na+ and water content as possible with ashing studies may still accurately quantitate excess tissue Na+ in these situations. Furthermore, when tissue [Na+] exceeds plasma [Na+], excess tissue Na+ must be present.

DSS-induced damage to basement membranes is repaired by matrix replacement and crosslinking.

Basement membranes are an ancient form of animal extracellular matrix. As important structural and functional components of tissues, basement membranes are subject to environmental damage and must be repaired while maintaining functions. Little is known about how basement membranes get repaired. This paucity stems from a lack of suitable in vivo models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult Drosophila DSS becomes incorporated into the basement membrane, promoting its expansion while decreasing its stiffness, which causes morphological changes to the underlying muscles. Remarkably, two days after withdrawal of DSS, the basement membrane is repaired by all measures of analysis. We used this new damage model to determine that repair requires collagen crosslinking and replacement of damaged components. Genetic and biochemical evidence indicates that crosslinking is required to stabilize the newly incorporated repaired Collagen IV rather than to stabilize the damaged Collagen IV. These results suggest that basement membranes are surprisingly dynamic.

Peroxidasin and eosinophil peroxidase, but not myeloperoxidase, contribute to renal fibrosis in the murine unilateral ureteral obstruction model.

Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.

Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes.

BACKGROUND: Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP. METHODS: We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score. RESULTS: We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease. CONCLUSIONS: Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.

Using Electrolyte Free Water Balance to Rationalize and Treat Dysnatremias.

Dysnatremias or abnormalities in plasma [Na+] are often termed disorders of water balance, an unclear physiologic concept often confused with changes in total fluid balance. However, most clinicians clearly recognize that hypertonic or hypotonic gains or losses alter plasma [Na+], while isotonic changes do not modify plasma [Na+]. This concept can be conceptualized as the electrolyte free water balance (EFWB), which defines the non-isotonic components of inputs and outputs to determine their effect on plasma [Na+]. EFWB is mathematically proportional to the rate of change in plasma [Na+] (dPNa/dt) and, therefore, is actively regulated to zero so that plasma [Na+] remains stable at its homeostatic set point. Dysnatremias are, therefore, disorders of EFWB and the relationship between EFWB and dPNa/dt provides a rationale for therapeutic strategies incorporating mass and volume balance. Herein, we leverage dPNa/dt as a desired rate of correction of plasma [Na+] to define a stepwise approach for the treatment of dysnatremias.

Blocking TGF-ß and ß-Catenin Epithelial Crosstalk Exacerbates CKD.

The TGF-ß and Wnt/ß-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF-ß has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF-ß signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF-ß type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF-ß receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF-ß receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/ß-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF-ß receptor impaired ß-catenin activity in vitro and in vivo Genetically restoring ß-catenin activity in proximal tubules lacking the TGF-ß receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF-ß receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of ß-catenin activity or an indirect effect of ß-catenin interacting with other growth factors. In conclusion, blocking TGF-ß and ß-catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.

Role of Hypohalous Acids in Basement Membrane Homeostasis.

SIGNIFICANCE: Basement membranes (BMs) are sheet-like structures of specialized extracellular matrix that underlie nearly all tissue cell layers including epithelial, endothelial, and muscle cells. BMs not only provide structural support but are also critical for the development, maintenance, and repair of organs. Animal heme peroxidases generate highly reactive hypohalous acids extracellularly and, therefore, target BMs for oxidative modification. Given the importance of BMs in tissue structure and function, hypohalous acid-mediated oxidative modifications of BM proteins represent a key mechanism in normal development and pathogenesis of disease. Recent Advances: Peroxidasin (PXDN), a BM-associated animal heme peroxidase, generates hypobromous acid (HOBr) to form sulfilimine cross-links within the collagen IV network of BM. These cross-links stabilize BM and are critical for animal tissue development. These findings highlight a paradoxical anabolic role for HOBr, which typically damages protein structure leading to dysfunction. CRITICAL ISSUES: The molecular mechanism whereby PXDN uses HOBr as a reactive intermediate to cross-link collagen IV, yet avoid collateral damage to nearby BM proteins, remains unclear. FUTURE DIRECTIONS: The exact identification and functional impact of specific hypohalous acid-mediated modifications of BM proteins need to be addressed to connect these modifications to tissue development and pathogenesis of disease. As seen with the sulfilimine cross-link of collagen IV, hypohalous acid oxidative events may be beneficial in select situations rather than uniformly deleterious. Antioxid. Redox Signal. 27, 839-854.