Increased activity of the metalloproteinase PAPP-A promotes diabetes-induced glomerular hypertrophy.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions. METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2. CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.

Independent of Renox, NOX5 Promotes Renal Inflammation and Fibrosis in Diabetes by Activating ROS-Sensitive Pathways.

Excessive production of renal reactive oxygen species (ROS) plays a major role in diabetic kidney disease (DKD). Here, we provide key findings demonstrating the predominant pathological role of the pro-oxidant enzyme NADPH oxidase 5 (NOX5) in DKD, independent of the previously characterized NOX4 pathway. In patients with diabetes, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors early growth response 1 (EGR-1), protein kinase C-α (PKC-α), and a key metabolic gene involved in redox balance, thioredoxin-interacting protein (TXNIP). In preclinical models of DKD, overexpression of NOX5 in Nox4-deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α, and PKC-ε. In addition, the only first-in-class NOX inhibitor, GKT137831, appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated upregulation of EGR1, PKC-α, and TXNIP induced by high glucose levels, as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared with other NOX isoforms such as NOX4, which may have been overinterpreted in previous rodent studies.

Adverse renal effects of NLRP3 inflammasome inhibition by MCC950 in an interventional model of diabetic kidney disease.

Activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome has been reported in diabetic complications including diabetic kidney disease (DKD). However, it remains unknown if NLRP3 inhibition is renoprotective in a clinically relevant interventional approach with established DKD. We therefore examined the effect of the NLRP3-specific inhibitor MCC950 in streptozotocin-induced diabetic mice to measure the impact of NLRP3 inhibition on renal inflammation and associated pathology in DKD. We identified an adverse effect of MCC950 on renal pathology in diabetic animals. Indeed, MCC950-treated diabetic animals showed increased renal inflammation and macrophage infiltration in association with enhanced oxidative stress as well as increased mesangial expansion and glomerulosclerosis when compared with vehicle-treated diabetic animals. Inhibition of the inflammasome by MCC950 in diabetic mice led to renal up-regulation of markers of inflammation (Il1ß, Il18 and Mcp1), fibrosis (Col1, Col4, Fn1, α-SMA, Ctgf and Tgfß1) and oxidative stress (Nox2, Nox4 and nitrotyrosine). In addition, enhanced glomerular accumulation of pro-inflammatory CD68 positive cells and pro-oxidant factor nitrotyrosine was identified in the MCC950-treated diabetic compared with vehicle-treated diabetic animals. Collectively, in this interventional model of established DKD, NLRP3 inhibition with MCC950 did not show renoprotective effects in diabetic mice. On the contrary, diabetic mice treated with MCC950 exhibited adverse renal effects particularly enhanced renal inflammation and injury including mesangial expansion and glomerulosclerosis.

The Complement Pathway: New Insights into Immunometabolic Signaling in Diabetic Kidney Disease.

Significance: The metabolic disorder, diabetes mellitus, results in microvascular complications, including diabetic kidney disease (DKD), which is partly believe to involve disrupted energy generation in the kidney, leading to injury that is characterized by inflammation and fibrosis. An increasing body of evidence indicates that the innate immune complement system is involved in the pathogenesis of DKD; however, the precise mechanisms remain unclear. Recent Advances: Complement, traditionally thought of as the prime line of defense against microbial intrusion, has recently been recognized to regulate immunometabolism. Studies have shown that the complement activation products, Complement C5a and C3a, which are potent pro-inflammatory mediators, can mediate an array of metabolic responses in the kidney in the diabetic setting, including altered fuel utilization, disrupted mitochondrial respiratory function, and reactive oxygen species generation. In diabetes, the lectin pathway is activated via autoreactivity toward altered self-surfaces known as danger-associated molecular patterns, or via sensing altered carbohydrate and acetylation signatures. In addition, endogenous complement inhibitors can be glycated, whereas diet-derived glycated proteins can themselves promote complement activation, worsening DKD, and lending support for environmental influences as an additional avenue for propagating complement-induced inflammation and kidney injury. Critical Issues: Recent evidence indicates that conventional renoprotective agents used in DKD do not target the complement, leaving this web of inflammatory stimuli intact. Future Directions: Future studies should focus on the development of novel pharmacological agents that target the complement pathway to alleviate inflammation, oxidative stress, and kidney fibrosis, thereby reducing the burden of microvascular diseases in diabetes. Antioxid. Redox Signal. 37, 781-801.

Fatal 3-Nitropropionic Acid Poisoning after Consuming Coconut Water.

We describe the fatal course of a patient with initial symptoms of vomiting and nausea who developed symptoms of dystonia, encephalopathy, and coma. The cause of death was poisoning with 3-nitropropionic acid from coconut water spoiled with the fungus Arthrinium saccharicola. We present the clinical findings and forensic analysis.

Effect of dipeptidyl peptidase-4 inhibitors on complement activation.

BACKGROUND: Adverse activation of the complement cascade in the innate immune system appears to be involved in development of vascular complications in diabetes. Dipeptidyl peptidase-4 (DPP-4) is a cell surface serine protease expressed in a variety of tissues. DPP-4 inhibitors are widely used in treatment of type 2 diabetes and appear to yield beneficial pleiotropic effects beyond their glucose-lowering action, for example, renoprotective and anti-inflammatory properties, but the exact mechanisms remain unknown. We hypothesised that DPP-4 inhibitors block adverse complement activation by inhibiting complement-activating serine proteases. MATERIALS AND METHODS: We analysed the effects of 7 different DPP-4 inhibitors on the lectin and classical pathway of the complement system in vitro by quantifying complement factor C4b deposition onto mannan or IgG coated surfaces, respectively. Furthermore, plasma concentrations of mannan-binding lectin (MBL), soluble membrane attack complex (sMAC), and C4b deposition were quantified in 71 patients with a recent acute coronary syndrome and glucose disturbances, randomly assigned to sitagliptin 100 mg (n = 34) or placebo (n = 37) for 12 weeks. RESULTS: All the 7 DPP-4 inhibitors tested in the study directly inhibited functional activity of the lectin pathway in a dose-dependent manner with varying potency in vitro. In vivo, MBL, sMAC, and C4b declined significantly during follow-up in both groups without significant effect of sitagliptin. CONCLUSIONS: We demonstrated an inhibitory effect of DPP-4 inhibitors on the lectin pathway in vitro. The clinical relevance of this effect of DPP-4 inhibitors remains to be fully elucidated.

A Single-Domain Antibody Targeting Complement Component C5 Acts as a Selective Inhibitor of the Terminal Pathway of the Complement System and Thus Functionally Mimicks the C-Terminal Domain of the Staphylococcus aureus SSL7 Protein.

The complement system is an efficient anti-microbial effector mechanism. On the other hand abnormal complement activation is involved in the pathogenesis of multiple inflammatory and hemolytic diseases. As general inhibition of the complement system may jeopardize patient health due to increased susceptibility to infections, the development of pathway-specific complement therapeutics has been a long-lasting goal over the last decades. In particular, pathogen mimicry has been considered as a promising approach for the design of selective anti-complement drugs. The C-terminal domain of staphylococcal superantigen-like protein 7 (SSL7), a protein secreted by Staphylococcus aureus, was recently found to be a specific inhibitor of the terminal pathway of the complement system, providing selective inhibition of cell lysis mediated by the membrane attack complex (MAC). We describe here the selection by phage display of a humanized single-domain antibody (sdAb) mimicking the C-terminal domain of SSL7. The antibody, called sdAb_E4, binds complement C5 with an affinity in the low micromolar range. Furthermore, sdAb_E4 induces selective inhibition of MAC-mediated lysis, allowing inhibition of red blood cell hemolysis and inhibition of complement deposition on apopto-necrotic cells, while maintaining efficient bactericidal activity of the complement terminal pathway. Finally, we present preliminary results indicating that sdAb_E4 may also be efficient in inhibiting hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria. Our data provide a proof of concept for the design of a selective MAC inhibitor capable of retaining complement bacteriolytic activity and this study opens up promising perspectives for the development of an sdAb_E4-derived therapeutics with application in the treatment of complement-mediated hemolytic disorders.

Increased all-cause mortality in patients with type 1 diabetes and high-expression mannan-binding lectin genotypes: a 12-year follow-up study.

OBJECTIVE: Mannan-binding lectin (MBL) is a complement-activating carbohydrate-recognizing molecule associated with diabetic nephropathy. MBL is associated with all-cause mortality in type 2 diabetes, but whether MBL is associated with mortality in type 1 diabetes remains unknown. We therefore aimed to investigate this. RESEARCH DESIGN AND METHODS: We studied an existing 12-year prospective cohort with type 1 diabetes with 198 patients with diabetic nephropathy (121 men, age 41 years [95% CI 40-42], estimated glomerular filtration rate [eGFR] 67 mL/min/1.73 m(2) [95% CI 63-70]) and 174 normoalbuminuric patients (103 men, age 43 years [95% CI 41-44], eGFR 93 mL/min/1.73 m(2) [95% CI 91-95]). Mortality rates were compared according to the concentration-determining MBL2 genotype or the MBL concentration. Patients were classified as having high or low MBL expression genotypes. The effect of MBL concentration was estimated by comparing patients with MBL concentrations above or below the median. RESULTS: Ninety-eight patients died during follow-up. The unadjusted hazard ratio (HR) for all-cause mortality was 1.61 (95% CI 1.07-2.43) for patients with high MBL expression genotypes versus patients with low MBL expression genotypes (P = 0.023). All-cause mortality was higher in patients with MBL concentrations above the median than in patients with MBL concentrations below the median (unadjusted HR 1.90 [95% CI 1.26-2.87], P = 0.002). CONCLUSIONS: High MBL expression genotypes and high MBL concentrations are both associated with increased mortality rates in type 1 diabetes compared with low MBL expression genotypes and low MBL concentrations.

Association of the pattern recognition molecule H-ficolin with incident microalbuminuria in an inception cohort of newly diagnosed type 1 diabetic patients: an 18 year follow-up study.

AIMS/HYPOTHESIS: Increasing evidence links complement activation through the lectin pathway to diabetic nephropathy. Adverse complement recognition of proteins modified by glycation has been suggested to trigger complement auto-attack in diabetes. H-ficolin (also known as ficolin-3) is a pattern recognition molecule that activates the complement cascade on binding to glycated surfaces, but the role of H-ficolin in diabetic nephropathy is unknown. We aimed to investigate the association between circulating H-ficolin levels and the incidence of microalbuminuria in type 1 diabetes. METHODS: We measured baseline H-ficolin levels and tracked the development of persistent micro- and macroalbuminuria in a prospective 18 year observational follow-up study of an inception cohort of 270 patients with newly diagnosed type 1 diabetes. RESULTS: Patients were followed for a median of 18 years (range 1-22 years). During follow-up, 75 patients developed microalbuminuria, defined as a persistent urinary albumin excretion rate (UAER) above 30 mg/24 h. When H-ficolin levels were divided into quartile groups an unadjusted Cox proportional hazards regression model showed a significant association with risk of incident microalbuminuria during follow-up (HR, fourth vs first quartile, 2.45; 95% CI 1.24, 4.85) (p = 0.01). This remained significant after adjusting for HbA1c, systolic blood pressure, smoking and baseline UAER (HR 2.09; 95% CI 1.03, 4.25) (p = 0.04). CONCLUSIONS/INTERPRETATION: Our data suggest that high levels of the complement activating molecule H-ficolin are associated with an increased risk of future progression to microalbuminuria in patients with newly diagnosed type 1 diabetes.

Association between endogenous complement inhibitor and myocardial salvage in patients with myocardial infarction.

BACKGROUND: Many pathogenic processes and diseases are the result of an erroneous activation of the complement cascade and a number of inhibitors of complement have thus been examined for anti-inflammatory actions. It was recently demonstrated that supraphysiological concentrations of the endogenous complement inhibitor MAp44 (also denoted MAP1) protect against myocardial reperfusion injury. In the present study, we examined the association between outcome after acute myocardial infarction (MI) and the plasma levels of MAp44 and its related proteins MASP-1 and MASP-3 in patients with first-time MI. In addition, we compared plasma levels of MAp44, MASP-1, and MASP-3 in MI patients to levels in a healthy control group. METHODS: A total of 192 MI patients and 140 control persons were included. Plasma samples were obtained and analysed with time-resolved immunofluorometric assays determining the plasma levels of MAp44, MASP-1, and MASP-3. The myocardial outcomes (salvage index and final infarct size) were measured by gated single-photon emission CT. RESULTS: MI patients had 18 % higher plasma levels of MAp44 (IQR 11-25%) as compared to the healthy control group (p<0.001. However, neither salvage index (Spearman rho -0.1, p=0.28) nor final infarct size (Spearman rho 0.02, p=0.83) correlated with plasma levels of MAp44. Likewise, MASP-1 and MASP-3 were elevated in MI patients (p=0.002 and p<0.001), but the levels were not correlated to outcome. CONCLUSIONS: Plasma levels of MAp44, MASP-1, and MASP-3 are significantly higher in patients with MI compared to healthy control persons, but are not associated with short-term outcome measured as salvage index and final infarct.

Glyoxalase-1 overexpression reduces endothelial dysfunction and attenuates early renal impairment in a rat model of diabetes.

AIMS/HYPOTHESIS: In diabetes, advanced glycation end-products (AGEs) and the AGE precursor methylglyoxal (MGO) are associated with endothelial dysfunction and the development of microvascular complications. In this study we used a rat model of diabetes, in which rats transgenically overexpressed the MGO-detoxifying enzyme glyoxalase-I (GLO-I), to determine the impact of intracellular glycation on vascular function and the development of early renal changes in diabetes. METHODS: Wild-type and Glo1-overexpressing rats were rendered diabetic for a period of 24 weeks by intravenous injection of streptozotocin. Mesenteric arteries were isolated to study ex vivo vascular reactivity with a wire myograph and kidneys were processed for histological examination. Glycation was determined by mass spectrometry and immunohistochemistry. Markers for inflammation, endothelium dysfunction and renal dysfunction were measured with ELISA-based techniques. RESULTS: Diabetes-induced formation of AGEs in mesenteric arteries and endothelial dysfunction were reduced by Glo1 overexpression. Despite the absence of advanced nephrotic lesions, early markers of renal dysfunction (i.e. increased glomerular volume, decreased podocyte number and diabetes-induced elevation of urinary markers albumin, osteopontin, kidney-inflammation-molecule-1 and nephrin) were attenuated by Glo1 overexpression. In line with this, downregulation of Glo1 in cultured endothelial cells resulted in increased expression of inflammation and endothelium dysfunction markers. In fully differentiated cultured podocytes incubation with MGO resulted in apoptosis. CONCLUSIONS/INTERPRETATION: This study shows that effective regulation of the GLO-I enzyme is important in the prevention of vascular intracellular glycation, endothelial dysfunction and early renal impairment in experimental diabetes. Modulating the GLO-I pathway therefore may provide a novel approach to prevent vascular complications in diabetes.

High altitude may alter oxygen availability and renal metabolism in diabetics as measured by hyperpolarized [1-(13)C]pyruvate magnetic resonance imaging.

The kidneys account for about 10% of the whole body oxygen consumption, whereas only 0.5% of the total body mass. It is known that intrarenal hypoxia is present in several diseases associated with development of kidney disease, including diabetes, and when renal blood flow is unaffected. The importance of deranged oxygen metabolism is further supported by deterioration of kidney function in patients with diabetes living at high altitude. Thus, we argue that reduced oxygen availability alters renal energy metabolism. Here, we introduce a novel magnetic resonance imaging (MRI) approach to monitor metabolic changes associated with diabetes and oxygen availability. Streptozotocin diabetic and control rats were given reduced, normal, or increased inspired oxygen in order to alter tissue oxygenation. The effects on kidney oxygen metabolism were studied using hyperpolarized [1-(13)C]pyruvate MRI. Reduced inspired oxygen did not alter renal metabolism in the control group. Reduced oxygen availability in the diabetic kidney altered energy metabolism by increasing lactate and alanine formation by 23% and 34%, respectively, whereas the bicarbonate flux was unchanged. Thus, the increased prevalence and severity of nephropathy in patients with diabetes at high altitudes may originate from the increased sensitivity toward inspired oxygen. This increased lactate production shifts the metabolic routs toward hypoxic pathways.