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.

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.

Mapping glycation and glycoxidation sites in collagen I of human cortical bone.

Accumulation of advanced glycation end products (AGEs), particularly in long-lived extracellular matrix proteins, has been implicated in pathogenesis of diabetic complications and in aging. Knowledge about specific locations of AGEs and their precursors within protein primary structure is critical for understanding their physiological and pathophysiological impact. However, the information on specific AGE sites is lacking. Here, we identified sequence positions of four major AGEs, carboxymethyllysine, carboxyethyllysine, 5-hydro-5-methyl imidazolone, and 5-hydro-imidazolone, and an AGE precursor fructosyllysine within the triple helical region of collagen I from cortical bone of human femurs. The presented map provides a basis for site-specific quantitation of AGEs and other non-enzymatic post-translational modifications and identification of those sites affected by aging, diabetes, and other diseases such as osteoporosis; it can also help in guiding future studies of AGE impact on structure and function of collagen I in bone.

Effect of ribose incubation on physical, chemical, and mechanical properties of human cortical bone.

Raman spectroscopy (RS) is sensitive to the accumulation of advanced glycation end-products (AGEs), and it measures matrix-sensitive properties that correlate with the fracture toughness of human cortical bone. However, it is unclear whether sugar-mediated accumulation of AGEs affects the fracture toughness of human cortical bone in a manner that is consistent with the negative correlations between amide I sub-peak ratios and fracture toughness. Upon machining 64 single-edge notched beam (SENB) specimens from cadaveric femurs (8 male and 7 female donors between 46 years and 61 years of age), pairs of SENB specimens were incubated in 15 mL of phosphate buffered saline with or without 0.1 M ribose for 4 weeks at 37 °C. After acquiring 10 Raman spectra per bone specimen (n = 32 per incubation group), paired SENB specimens were loaded in three-point bending at a quasi-static or a high loading rate approximating 10-4 s-1 or 10-2 s-1, respectively (n = 16 per incubation group per loading rate). While 2 amide I sub-peak ratios, I1670/I1640 and I1670/I1610, decreased by 3-5% with a 100% increase in AGE content, as confirmed by fluorescence measurements, the ribose incubation to accumulate AGEs in bone did not affect linear elastic (KIc) nor non-linear elastic (KJc) measurements of bone's ability to resist crack growth. Moreover, AGE accumulation did not affect the change in these properties when the loading rate changed. Increasing the loading rate increased KIc but decreased KJc. Ribose incubation did not affect mineral-related RS properties such as mineral-to-matrix ratios, Type B carbonate substitutions, and crystallinity. It did however increase the thermal stability of demineralized bone (differential scanning calorimetry), without affecting the network connectivity of the organic matrix (i.e., maximum slope during a hydrothermal isometric tension test of demineralized bone). In conclusion, RS is sensitive to AGE accumulation via the amide I band (plus the hydroxyproline-to-proline ratio), but the increase in AGE content due to ribose incubation was not sufficient to affect the fracture toughness of human cortical bone.

Causative or associative: A critical review of the role of advanced glycation end-products in bone fragility.

The accumulation of advanced glycation end-products (AGEs) in the organic matrix of bone with aging and chronic disease such as diabetes is thought to increase fracture risk independently of bone mass. However, to date, there has not been a clinical trial to determine whether inhibiting the accumulation of AGEs is effective in preventing low-energy, fragility fractures. Moreover, unlike with cardiovascular or kidney disease, there are also no pre-clinical studies demonstrating that AGE inhibitors or breakers can prevent the age- or diabetes-related decrease in the ability of bone to resist fracture. In this review, we critically examine the case for a long-standing hypothesis that AGE accumulation in bone tissue degrades the toughening mechanisms by which bone resists fracture. Prior research into the role of AGEs in bone has primarily measured pentosidine, an AGE crosslink, or bulk fluorescence of hydrolysates of bone. While significant correlations exist between these measurements and mechanical properties of bone, multiple AGEs are both non-fluorescent and non-crosslinking. Since clinical studies are equivocal on whether circulating pentosidine is an indicator of elevated fracture risk, there needs to be a more complete understanding of the different types of AGEs including non-crosslinking adducts and multiple non-enzymatic crosslinks in bone extracellular matrix and their specific contributions to hindering fracture resistance (biophysical and biological). By doing so, effective strategies to target AGE accumulation in bone with minimal side effects could be investigated in pre-clinical and clinical studies that aim to prevent fragility fractures in conditions that bone mass is not the underlying culprit.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

Collagen IVα345 dysfunction in glomerular basement membrane diseases. II. Crystal structure of the α345 hexamer.

Our recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture's disease (GP) and Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this "Zurich" variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer-trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions.

Post-translational modifications in collagen type I of bone in a mouse model of aging.

The fracture resistance of cortical bone and matrix hydration are known to decline with advanced aging. However, the underlying mechanisms remain poorly understood, and so we investigated levels of matrix proteins and post-translational modifications (PTM) of collagen I in extracts from the tibia of 6-mo. and 20-mo. old BALB/c mice (female and male analysis done separately). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the levels of collagen I deamidation at specific asparagine (Asn) and glutamine (Gln) residues significantly increased with age. Other non-enzymatic PTMs such as carboxymethylation of lysine (CML) were detected as well, but the relative abundance did not vary with age. No significant age-related differences in the abundance of hydroxylysine glycosylation sites were found, but hydroxylation levels at a few of the numerous lysine and proline hydroxylation sites significantly changed by a small amount with age. We performed molecular modeling and dynamics (MD) simulations for three triple helical fragments representing collagen I regions with prominent age-dependent increases in deamidation as identified by LC-MS/MS of male extracts. These 3 fragments included deamidated Asn and Gln residues as follows: 1) an Asn428 site of the α2(I) chain in which deamidation levels increased from 4.4% at 6-mo. to 8.1% at 20-mo., 2) an Asn983 site of the α2(I) chain with a deamidation increase from 18.3% to 36.8% with age and an Asn1052 site of the α1(I) chain with consistent deamidation levels of ~60% across the age groups, and 3) a Gln410 site of the α1(I) chain that went from no detectable deamidation at 6-mo. to 2.7% at 20-mo. and a neighboring Asn421 site of the same chain with an age-related deamidation increase from 3.6% to 16.3%. The deamidation levels at these sites inversely correlated with an estimate of toughness determined from three-point bending tests of the femur mid-diaphysis. MD revealed that the sidechains become more negatively charged at deamidated sites and that deamidation alters hydrogen bonding with water along the collagen backbone while increasing water interactions with the aspartic and glutamic acid sidechains. Our findings suggest a new mechanism of the age-dependent reduction in the fracture resistance of cortical bone whereby deamidation of Asn and Glu residues redistributes bound water within collagen I triple helix.

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.

Manipulating the Amount and Structure of the Organic Matrix Affects the Water Compartments of Human Cortical Bone.

Being predictors of the mechanical properties of human cortical bone, bound and pore water measurements by magnetic resonance (MR) imaging are being developed for the clinical assessment of fracture risk. While pore water is a surrogate of cortical bone porosity, the determinants of bound water are unknown. Manipulation of organic matrix properties by oxidative deproteinization, thermal denaturation, or nonenzymatic glycation lowers bone toughness. Because bound water contributes to bone toughness, we hypothesized that each of these matrix manipulations affect bound water fraction (Vbw/Vbone). Immersing cadaveric bone samples in sodium hypochlorite (NaClO) for 96 hours did not affect tissue mineral density or cortical porosity, but rather decreased Vbw/Vbone and increased short-T2 pore water signals as determined by 1H nuclear MR relaxometry (1H NMR). Moreover, the post treatment Vbw/Vbone linearly correlated with the remaining weight fraction of the organic matrix. Heating bone samples at 110°C, 120°C, 130°C, and then 140°C (∼24 hours per temperature and rehydration for ∼24 hours before 1H NMR analysis) did not affect Vbw/Vbone. After subsequently heating them at 200°C, Vbw/Vbone increased. Boiling bone samples followed by heating at 110°C, 120°C, and then 130°C in water under pressure (8 hours per temperature) had a similar effect on Vbw/Vbone. Raman spectroscopy analysis confirmed that the increase in Vbw/Vbone coincided with an increase in an Amide I subpeak ratio that is sensitive to changes in the helical structure of collagen I. Glycation of bone by ribose for 4 weeks, but not in glucose for 16 weeks, decreased Vbw/Vbone, although the effect was less pronounced than that of oxidative deproteinization or thermal denaturation. We propose that MR measurements of bound water reflect the amount of bone organic matrix and can be modulated by collagen I helicity and by sugar-derived post translational modifications of the matrix. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Two Specific Sulfatide Species Are Dysregulated during Renal Development in a Mouse Model of Alport Syndrome.

Alport syndrome is caused by mutations in collagen IV that alter the morphology of renal glomerular basement membrane. Mutations result in proteinuria, tubulointerstitial fibrosis, and renal failure but the pathogenic mechanisms are not fully understood. Using imaging mass spectrometry, we aimed to determine whether the spatial and/or temporal patterns of renal lipids are perturbed during the development of Alport syndrome in the mouse model. Our results show that most sulfatides are present at similar levels in both the wild-type (WT) and the Alport kidneys, with the exception of two specific sulfatide species, SulfoHex-Cer(d18:2/24:0) and SulfoHex-Cer(d18:2/16:0). In the Alport but not in WT kidneys, the levels of these species mirror the previously described abnormal laminin expression in Alport syndrome. The presence of these sulfatides in renal tubules but not in glomeruli suggests that this specific aberrant lipid pattern may be related to the development of tubulointerstitial fibrosis in Alport disease.

Changes in urinary metabolome related to body fat involve intermediates of choline processing by gut microbiota.

Countering the obesity pandemic will require better understanding of disease mechanisms and development of new diagnostic methods. Small molecule metabolites excreted in urine can be important biomarkers of disease progression and treatment. However, with multiple pathways involved, it has been challenging to identify key pathway(s) that closely follow disease features such as body fat. We employed a high-fat diet (HFD) mouse model of obesity with the goal of determining changes in urinary metabolite profile related to body fat using proton nuclear magnetic resonance (1H NMR). Several urinary metabolites with significantly lower levels in HFD compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle, amino acid, nicotinamide, and choline metabolism including 2-hydroxydlutarate, cis-aconitate, trans-aconitate, alanine, creatine, trigonelline, dimethylamine, and trimethylamine. However, levels of only two metabolites, namely dimethylamine and trimethylamine, showed significant reverse correlation with total body fat. These metabolites derive from choline processing by gut microbiota and may be prospective biomarkers indicative of accumulation of body fat in obesity.

Imaging mass spectrometry reveals direct albumin fragmentation within the diabetic kidney.

Albumin degradation in the renal tubules is impaired in diabetic nephropathy such that levels of the resulting albumin fragments increase with the degree of renal injury. However, the mechanism of albumin degradation is unknown. In particular, fragmentation of the endogenous native albumin has not been demonstrated in the kidney and the enzymes that may contribute to fragmentation have not been identified. To explore this we utilized matrix-assisted laser desorption/ionization imaging mass spectrometry for molecular profiling of specific renal regions without disturbing distinct tissue morphology. Changes in protein expression were measured in kidney sections of eNOS-/-db/db mice, a model of diabetic nephropathy, by high spatial resolution imaging allowing molecular localizations at the level of single glomeruli and tubules. Significant increases were found in the relative abundances of several albumin fragments in the kidney of the mice with diabetic nephropathy compared with control nondiabetic mice. The relative abundance of fragments detected correlated positively with the degree of nephropathy. Furthermore, specific albumin fragments accumulating in the lumen of diabetic renal tubules were identified and predicted the enzymatic action of cathepsin D based on cleavage specificity and in vitro digestions. Importantly, this was demonstrated directly in the renal tissue with the endogenous nonlabeled murine albumin. Thus, our results provide molecular insights into the mechanism of albumin degradation in diabetic nephropathy.

Halogens are key cofactors in building of collagen IV scaffolds outside the cell.

PURPOSE OF REVIEW: The purpose of this review is to highlight recent advances in understanding the molecular assembly of basement membranes, as exemplified by the glomerular basement membrane (GBM) of the kidney filtration apparatus. In particular, an essential role of halogens in the basement membrane formation has been discovered. RECENT FINDINGS: Extracellular chloride triggers a molecular switch within non collagenous domains of collagen IV that induces protomer oligomerization and scaffold assembly outside the cell. Moreover, bromide is an essential cofactor in enzymatic cross-linking that reinforces the stability of scaffolds. Halogenation and halogen-induced oxidation of the collagen IV scaffold in disease states damage scaffold function. SUMMARY: Halogens play an essential role in the formation of collagen IV scaffolds of basement membranes. Pathogenic damage of these scaffolds by halogenation and halogen-induced oxidation is a potential target for therapeutic interventions.

Assessing glycation-mediated changes in human cortical bone with Raman spectroscopy.

Establishing a non-destructive method for spatially assessing advanced glycation end-products (AGEs) is a potentially useful step toward investigating the mechanistic role of AGEs in bone quality. To test the hypothesis that the shape of the amide I in the Raman spectroscopy (RS) analysis of bone matrix changes upon AGE accumulation, we incubated paired cadaveric cortical bone in ribose or glucose solutions and in control solutions for 4 and 16 weeks, respectively, at 37°C. Acquiring 10 spectra per bone with a 20X objective and a 830 nm laser, RS was sensitive to AGE accumulation (confirmed by biochemical measurements of pentosidine and fluorescent AGEs). Hyp/Pro ratio increased upon glycation using either 0.1 M ribose, 0.5 M ribose or 0.5 M glucose. Glycation also decreased the amide I sub-peak ratios (cm-1 ) 1668/1638 and 1668/1610 when directly calculated using either second derivative spectrum or local maxima of difference spectrum, though the processing method (eg, averaged spectrum vs individual spectra) to minimize noise influenced detection of differences for the ribose-incubated bones. Glycation however did not affect these sub-peak ratios including the matrix maturity ratio (1668/1690) when calculated using indirect sub-band fitting. The amide I sub-peak ratios likely reflected changes in the collagen I structure.

Low bone toughness in the TallyHO model of juvenile type 2 diabetes does not worsen with age.

Fracture risk increases as type 2 diabetes (T2D) progresses. With the rising incidence of T2D, in particular early-onset T2D, a representative pre-clinical model is needed to study mechanisms for treating or preventing diabetic bone disease. Towards that goal, we hypothesized that fracture resistance of bone from diabetic TallyHO mice decreases as the duration of diabetes increases. Femurs and lumbar vertebrae were harvested from male, TallyHO mice and male, non-diabetic SWR/J mice at 16weeks (n≥12 per strain) and 34weeks (n≥13 per strain) of age. As is characteristic of this model of juvenile T2D, the TallyHO mice were obese and hyperglycemic at an early age (5weeks and 10weeks of age, respectively). The femur mid-shaft of TallyHO mice had higher tissue mineral density and larger cortical area, as determined by micro-computed tomography, compared to the femur mid-shaft of SWR/J mice, irrespective of age. As such, the diabetic rodent bone was structurally stronger than the non-diabetic rodent bone, but the higher peak force endured by the diaphysis during three-point (3pt) bending was not independent of the difference in body weight. Upon accounting for the structure of the femur diaphysis, the estimated toughness at 16weeks and 34weeks was lower for the diabetic mice than for non-diabetic controls, but neither toughness nor estimated material strength and resistance to crack growth (3pt bending of contralateral notched femur) decreased as the duration of hyperglycemia increased. With respect to trabecular bone, there were no differences in the compressive strength of the L6 vertebral strength between diabetic and non-diabetic mice at both ages despite a lower trabecular bone volume for the TallyHO than for the SWR/J mice at 34weeks. Amide I sub-peak ratios as determined by Raman Spectroscopy analysis of the femur diaphysis suggested a difference in collagen structure between diabetic and non-diabetic mice, although there was not a significant difference in matrix pentosidine between the groups. Overall, the fracture resistance of bone in the TallyHO model of T2D did not progressively decrease with increasing duration of hyperglycemia. However, given the variability in hyperglycemia in this model, there were correlations between blood glucose levels and certain structural properties including peak force.

Changes in the Fracture Resistance of Bone with the Progression of Type 2 Diabetes in the ZDSD Rat.

Individuals with type 2 diabetes (T2D) have a higher fracture risk compared to non-diabetics, even though their areal bone mineral density is normal to high. Identifying the mechanisms whereby diabetes lowers fracture resistance requires well-characterized rodent models of diabetic bone disease. Toward that end, we hypothesized that bone toughness, more so than bone strength, decreases with the duration of diabetes in ZDSD rats. Bones were harvested from male CD(SD) control rats and male ZDSD rats at 16 weeks (before the onset of hyperglycemia), at 22 weeks (5-6 weeks of hyperglycemia), and at 29 weeks (12-13 weeks of hyperglycemia). There were at least 12 rats per strain per age group. At 16 weeks, there was no difference in either body weight or glucose levels between the two rat groups. Within 2 weeks of switching all rats to a diet with 48 % of kcal from fat, only the ZDSD rats developed hyperglycemia (>250 mg/dL). They also began to lose body weight at 21 weeks. CD(SD) rats remained normoglycemic (<110 mg/dL) on the high-fat diet and became obese (>600 g). From micro-computed tomography (µCT) analysis of a lumbar vertebra and distal femur, trabecular bone volume did not vary with age among the non-diabetic rats but was lower at 29 weeks than at 16 weeks or at 22 weeks for the diabetic rats. Consistent with that finding, µCT-derived intra-cortical porosity (femur diaphysis) was higher for ZDSD following ~12 weeks of hyperglycemia than for age-matched CD(SD) rats. Despite an age-related increase in mineralization in both rat strains (µCT and Raman spectroscopy), material strength of cortical bone (from three-point bending tests) increased with age only in the non-diabetic CD(SD) rats. Moreover, two other material properties, toughness (radius) and fracture toughness (femur), significantly decreased with the duration of T2D in ZDSD rats. This was accompanied by the increase in the levels of the pentosidine (femur). However, pentosidine was not significantly higher in diabetic than in non-diabetic bone at any time point. The ZDSD rat, which has normal leptin signaling and becomes diabetic after skeletal maturity, provides a pre-clinical model of diabetic bone disease, but a decrease in body weight during prolonged diabetes and certain strain-related differences before the onset of hyperglycemia should be taken into consideration when interpreting diabetes-related differences.

Pyridoxamine reduces postinjury fibrosis and improves functional recovery after acute kidney injury.

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.

Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful molecular mapping technology that offers unbiased visualization of the spatial arrangement of biomolecules in tissue. Although there has been a significant increase in the number of applications employing this technology, the extracellular matrix (ECM) has received little attention, likely because ECM proteins are mostly large, insoluble and heavily cross-linked. We have developed a new sample preparation approach to enable MALDI IMS analysis of ECM proteins in tissue. Prior to freezing and sectioning, intact tissues are decellularized by incubation in sodium dodecyl sulfate. Decellularization removes the highly abundant, soluble species that dominate a MALDI IMS spectrum while preserving the structural integrity of the ECM. In situ tryptic hydrolysis and imaging of tryptic peptides are then carried out to accommodate the large sizes of ECM proteins. This new approach allows the use of MALDI IMS for identification of spatially specific changes in ECM protein expression and modification in tissue.

Pyridoxamine protects proteins from damage by hypohalous acids in vitro and in vivo.

Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes 64:2242-2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy.