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Krüppel-like factors (KLFs) are transcription factors with important roles in tissue homeostasis. KLF4 possesses antithrombotic and anti-inflammatory properties. In this issue, Estrada et al. show that endothelial KLF4 prevents complement deposition in glomeruli and in its absence the cell-bound complement regulator CD55 was reduced. The study included endothelial-specific KLF4 knockdown mice that mimic thrombotic microangiopathy and thrombotic microangiopathy patient biopsies showing decreased KLF4 and CD55. The results suggest that KLF4 is involved in the regulation of glomerular complement deposition.

During severe inflammatory and infectious diseases, various mediators modulate the equilibrium of vascular tone, inflammation, coagulation and thrombosis. This Review describes the interactive roles of the renin–angiotensin system, the complement system, and the closely linked kallikrein–kinin and contact systems in cell biological functions such as vascular tone and leakage, inflammation, chemotaxis, thrombosis and cell proliferation. Specific attention is given to the role of these systems in systemic inflammation in the vasculature and tissues during hereditary angioedema, cardiovascular and renal glomerular disease, vasculitides and COVID-19. Moreover, we discuss the therapeutic implications of these complex interactions, given that modulation of one system may affect the other systems, with beneficial or deleterious consequences., The renin–angiotensin, complement and kallikrein–kinin systems comprise a multitude of mediators that modulate physiological responses during inflammatory and infectious diseases. This Review investigates the complex interactions between these systems and how these are dysregulated in various conditions, including cardiovascular diseases and COVID-19, as well as their therapeutic implications.

A recently developed inhibitor of retrograde transport, namely Retro-2.1, proved to be a potent and broad-spectrum lead in vitro against intracellular pathogens, such as toxins, parasites, intracellular bacteria and viruses. To circumvent its low aqueous solubility, a formulation in poly(ethylene glycol)-block-poly(D,L)lactide micelle nanoparticles was developed. This formulation enabled the study of the pharmacokinetic parameters of Retro-2.1 in mice following intravenous and intraperitoneal injections, revealing a short blood circulation time, with an elimination half-life of 5 and 6.7 h, respectively. To explain the poor pharmacokinetic parameters, the metabolic stability of Retro-2.1 was studied in vitro and in vivo, revealing fast cytochrome-P-450-mediated metabolism into a less potent hydroxylated analogue. Subcutaneous injection of Retro-2.1 formulated in a biocompatible and bioresorbable polymer-based thermosensitive hydrogel allowed for sustained release of the drug, with an elimination half-life of 19 h, and better control of its metabolism. This study provides a guideline on how to administer this promising lead in vivo in order to study its efficacy.

Extracellular vesicles can mediate cell-to-cell communication, or relieve the parent cell of harmful substances, in order to maintain cellular integrity. The content of extracellular vesicles includes miRNAs, mRNAs, growth factors, complement factors, cytokines, chemokines and receptors. These may contribute to inflammatory and infectious diseases by the exposure or transfer of potent effectors that induce vascular inflammation by leukocyte recruitment and thrombosis. Furthermore, vesicles release cytokines and induce their release from cells. Extracellular vesicles possess immune modulatory and anti-microbial properties, and induce receptor signaling in the recipient cell, not least by the transfer of pro-inflammatory receptors. Additionally, the vesicles may carry virulence factors systemically. Extracellular vesicles in blood and urine can contribute to the development of kidney diseases or exhibit protective effects. In this review we will describe the role of EVs in inflammation, thrombosis, immune modulation, angiogenesis, oxidative stress, renal tubular regeneration and infection. Furthermore, we will delineate their contribution to renal ischemia/reperfusion, vasculitis, glomerulonephritis, lupus nephritis, thrombotic microangiopathies, IgA nephropathy, acute kidney injury, urinary tract infections and renal transplantation. Due to their content of miRNAs and growth factors, or when loaded with nephroprotective modulators, extracellular vesicles have the potential to be used as therapeutics for renal regeneration.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury., Donor-specific antibodies in sensitized recipients may cause kidney transplant rejection. Here the authors show that complement component C3 inhibition prolongs graft survival by inhibiting T and B cell proliferation/activation and hence tissue injury, despite antibody levels remaining unaffected.

Shiga toxin-producing

Microvesicles are shed from cell surfaces during infectious or inflammatory conditions and may contribute to the pathogenesis of disease. During Shiga toxin-producing Escherichia coli (STEC) infection, microvesicles are released from blood cells. These microvesicles play a part in inflammation, thrombosis, hemolysis, and the transfer of the main virulence factor of STEC strains, Shiga toxin, to target organ cells. This chapter describes how to isolate blood cell- and cell culture-derived microvesicles from plasma or cell culture medium, respectively, and how to characterize these microvesicles by various methods, with special focus on Shiga toxin-associated microvesicles.

Extracellular vesicles are cell-derived membrane particles ranging from 30 to 5,000 nm in size, including exosomes, microvesicles, and apoptotic bodies. They are released under physiological conditions, but also upon cellular activation, senescence, and apoptosis. They play an important role in intercellular communication. Their release may also maintain cellular integrity by ridding the cell of damaging substances. This review describes the biogenesis, uptake, and detection of extracellular vesicles in addition to the impact that they have on recipient cells, focusing on mechanisms important in the pathophysiology of kidney diseases, such as thrombosis, angiogenesis, tissue regeneration, immune modulation, and inflammation. In kidney diseases, extracellular vesicles may be utilized as biomarkers, as they are detected in both blood and urine. Furthermore, they may contribute to the pathophysiology of renal disease while also having beneficial effects associated with tissue repair. Because of their role in the promotion of thrombosis, inflammation, and immune-mediated disease, they could be the target of drug therapy, whereas their favorable effects could be utilized therapeutically in acute and chronic kidney injury.

Adequate cleavage of von Willebrand factor (VWF) prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM) was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3), cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure., Highlights • Neutrophil proteases in the glomerular basement membrane cleave VWF and may protect the kidney from microthrombi. • VWF cleavage would be activated by neutrophil influx and compensate for the prothrombotic mechanisms during inflammation. • This mechanism may compensate for lack of ADAMTS13 and explain why TTP patients seldom develop end-stage renal failure. The study demonstrates a mechanism by which the kidney is protected from blood clotting during inflammation. In the inflammatory setting white blood cells infiltrate tissues. In this study we showed that enzymes released from white blood cells into the kidney decrease the size of blood clots. This is a general mechanism but could also explain why patients with thrombotic thrombocytopenic purpura, who develop widespread blood clots in many organs, do not usually develop severe kidney failure.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

Orphan drugs designed to treat rare diseases are often overpriced per patient. Novel treatments are sometimes even more expensive for patients with ultra-rare diseases, in part due to the limited number of patients. Pharmaceutical companies that develop a patented life-saving drug are in a position to charge a very high price, which, at best, may enable these companies to further develop drugs for use in rare disease. However, is there a limit to how much a life-saving drug should cost annually per patient? Government interventions and regulations may opt to withhold a life-saving drug solely due to its high price and cost-effectiveness. Processes related to drug pricing, reimbursement, and thereby availability, vary between countries, thus having implications on patient care. These processes are discussed, with specific focus on three drugs used in pediatric nephrology: agalsidase beta (for Fabry disease), eculizumab (for atypical hemolytic uremic syndrome), and cysteamine bitartrate (for cystinosis). Access to and costs of orphan drugs have most profound implications for patients, but also for their physicians, hospitals, insurance policies, and society at large, particularly from financial and ethical standpoints.

Complement is activated during Shiga toxin–producing Escherichia coli –associated hemolytic uremic syndrome (STEC-HUS). There is evidence of complement activation via the alternative pathway in STEC-HUS patients as well as from in vivo and in vitro models. Ozaki et al. demonstrate activation of the mannose-binding lectin (MBL) pathway in Shiga toxin–treated mice expressing human MBL2, but lacking murine Mbl s. Treatment with anti–human MBL2 antibody was protective, suggesting that MBL pathway activation also contributes to Shiga toxin–mediated renal injury.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1k and P2k phenotype RBCs, expressing high levels of the Pk Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.

Shiga toxin is the main virulence factor of enterohemorrhagic Escherichia coli, a non-invasive pathogen that releases virulence factors in the intestine, causing hemorrhagic colitis and, in severe cases, hemolytic uremic syndrome (HUS). HUS manifests with acute renal failure, hemolytic anemia and thrombocytopenia. Shiga toxin induces endothelial cell damage leading to platelet deposition in thrombi within the microvasculature and the development of thrombotic microangiopathy, mostly affecting the kidney. Red blood cells are destroyed in the occlusive capillary lesions. This review focuses on the importance of microvesicles shed from blood cells and their participation in the prothrombotic lesion, in hemolysis and in the transfer of toxin from the circulation into the kidney. Shiga toxin binds to blood cells and may undergo endocytosis and be released within microvesicles. Microvesicles normally contribute to intracellular communication and remove unwanted components from cells. Many microvesicles are prothrombotic as they are tissue factor- and phosphatidylserine-positive. Shiga toxin induces complement-mediated hemolysis and the release of complement-coated red blood cell-derived microvesicles. Toxin was demonstrated within blood cell-derived microvesicles that transported it to renal cells, where microvesicles were taken up and released their contents. Microvesicles are thereby involved in all cardinal aspects of Shiga toxin-associated HUS, thrombosis, hemolysis and renal failure.

Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.

The kinin system is activated during vasculitis and may contribute to chronic inflammation. C1-inhibitor is the main inhibitor of the kinin system. In this study, we investigated the presence of the kinin B1 receptor on endothelial microvesicles and its contribution to the inflammatory process. Compared with controls (n=15), patients with acute vasculitis (n=12) had markedly higher levels of circulating endothelial microvesicles, identified by flow cytometry analysis, and significantly more microvesicles that were positive for the kinin B1 receptor (P

13 p.-3 fig. Goodship, Timothy H.J. et al., In both atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) complement plays a primary role in disease pathogenesis. Herein we report the outcome of a 2015 Kidney Disease: Improving Global Outcomes (KDIGO)Controversies Conference where key issues in the management of these 2 diseases were considered by a global panel of experts. Areas addressed included renal pathology, clinical phenotype and assessment, genetic drivers of disease, acquired drivers of disease, and treatment strategies. In order to help guide clinicians who are caring for such patients, recommendations for best treatment strategies were discussed at length, providing the evidence base underpinning current treatment options. Knowledge gaps were identified and a prioritized research agenda was proposed to resolve outstanding controversial issues., The conference was sponsored by Kidney Disease: Improving Global Outcomes (KDIGO) and supported in part by unrestricted educational grants from Achillion Pharmaceuticals, Akari Therapeutics, Alexion Pharmaceuticals, and Omeros.

Hemolytic uremic syndrome, a life-threatening disease often accompanied by acute renal failure, usually occurs after gastrointestinal infection with Shiga toxin 2 (Stx2)-producing Escherichia coli. Stx2 binds to the glycosphingolipid globotriaosylceramide receptor, expressed by renal epithelial cells, and triggers apoptosis by activating the apoptotic factor Bax. Signaling via the ouabain/Na,K-ATPase/IP3R/NF-κB pathway increases expression of Bcl-xL, an inhibitor of Bax, suggesting that ouabain might protect renal cells from Stx2-triggered apoptosis. Here, exposing rat proximal tubular cells to Stx2 in vitro resulted in massive apoptosis, upregulation of the apoptotic factor Bax, increased cleaved caspase-3, and downregulation of the survival factor Bcl-xL; co-incubation with ouabain prevented all of these effects. Ouabain activated the NF-κB antiapoptotic subunit p65, and the inhibition of p65 DNA binding abolished the antiapoptotic effect of ouabain in Stx2-exposed tubular cells. Furthermore, in vivo, administration of ouabain reversed the imbalance between Bax and Bcl-xL in Stx2-treated mice. Taken together, these results suggest that ouabain can protect the kidney from the apoptotic effects of Stx2.

Item does not contain fulltext Atypical hemolytic uremic syndrome (aHUS) emerged during the last decade as a disease largely of complement dysregulation. This advance facilitated the development of novel, rational treatment options targeting terminal complement activation, e.g., using an anti-C5 antibody (eculizumab). We review treatment and patient management issues related to this therapeutic approach. We present consensus clinical practice recommendations generated by HUS International, an international expert group of clinicians and basic scientists with a focused interest in HUS. We aim to address the following questions of high relevance to daily clinical practice: Which complement investigations should be done and when? What is the importance of anti-factor H antibody detection? Who should be treated with eculizumab? Is plasma exchange therapy still needed? When should eculizumab therapy be initiated? How and when should complement blockade be monitored? Can the approved treatment schedule be modified? What approach should be taken to kidney and/or combined liver-kidney transplantation? How should we limit the risk of meningococcal infection under complement blockade therapy? A pressing question today regards the treatment duration. We discuss the need for prospective studies to establish evidence-based criteria for the continuation or cessation of anticomplement therapy in patients with and without identified complement mutations.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC ( E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.

Hemolytic uremic syndrome (HUS) is commonly associated with Shiga toxin (Stx)–producing Escherichia coli O157:H7 infection. This study examined patient samples for complement activation on leukocyte-platelet complexes and microparticles, as well as donor samples for Stx and lipopolysaccharide (O157LPS)–induced complement activation on platelet-leukocyte complexes and microparticles. Results, analyzed by flow cytometry, showed that whole blood from a child with HUS had surface-bound C3 on 30% of platelet-monocyte complexes compared with 14% after recovery and 12% in pediatric controls. Plasma samples from 12 HUS patients were analyzed for the presence of microparticles derived from platelets, monocytes, and neutrophils. Acute-phase samples exhibited high levels of platelet microparticles and, to a lesser extent, monocyte microparticles, both bearing C3 and C9. Levels decreased significantly at recovery. Stx or O157LPS incubated with donor whole blood increased the population of platelet-monocyte and platelet-neutrophil complexes with surface-bound C3 and C9, an effect enhanced by costimulation with Stx and O157LPS. Both Stx and O157LPS induced the release of C3- and C9-bearing microparticles from platelets and monocytes. Released microparticles were phagocytosed by neutrophils. The presence of complement on platelet-leukocyte complexes and microparticles derived from these cells suggests a role in the inflammatory and thrombogenic events that occur during HUS.

Vasculitis is a systemic autoimmune inflammatory disease, characterized by inflammation in and around vessel walls leading to perturbed vessel patency and tissue damage. Many different organs may be involved. In this review pathogenetic mechanisms of vasculitis are discussed, with special reference to activation of the kinin system. Mechanisms of kinin system activation are described ultimately leading to release of kinins from high-molecular-weight-kininogen. These vasoactive peptides promote inflammation. Conclusion: Kinin system activation during vasculitis promotes inflammation.

BACKGROUND: Renal glomerular filtration rate (GFR) of pediatric cancer patients at diagnosis has previously been investigated in a limited number of studies. PROCEDURE: GFR, measured by iohexol clearance, was prospectively investigated in 55 children over the age of 1 year with malignancies, (group A). Elevated GFR (>175 ml/min/1.73 m(2)) at diagnosis was found. To investigate if this finding was consistent, a second group of 76 children with malignancies was studied, (group B). As a method control for GFR obtained by iohexol clearance, group A and B together were compared to 298 pediatric patients without cancer, group C. RESULTS: GFR was elevated in 40/131 (31%) in Group A + B but only in 17/298 (6%) in Group C. GFR was significantly higher in children aged 1-5 in group A + B (47%) compared to group C (3%). Bone marrow involvement was significantly associated with higher GFR. Children without bone marrow involvement also hyperfiltrated more often than controls, but less often. Urea in urine was used as a marker of renal protein clearance in 14 patients in group A. A significant correlation between u-urea (mmol/L)/u-creatinine (mmol/L) and GFR was noted. CONCLUSIONS: Hyperfiltration is sometimes present in children with cancer at diagnosis. This may be related to increased amino acid turn over in patients with a large tumor burden. An elevated initial GFR in a child with cancer, which normalizes after chemotherapy may indicate chemotherapy-induced decreased renal function, but can be due to normalization of an initially high GFR. Pediatr Blood Cancer © 2011 Wiley-Liss, Inc. (Less)

Most patients with IgA nephropathy exhibit complement deposition in the glomerular mesangium. Certain cases of IgA nephropathy have been associated with reduced levels of complement factor H. A recent study could not demonstrate mutations at the C-terminal of factor H. We describe a novel heterozygous mutation in factor H, position A48S (nucleotide position 142 G > T, alanine > serine), detected in exon 2 of a 14-year-old girl with IgA nephropathy. The patient exhibited reduced levels of C3 and factor H, the latter suggesting that the mutation affected factor H secretion. The patient developed initial signs and symptoms of glomerulonephritis at the age of 9 years but presented again at the age of 14 years with weight gain, renal failure, nephrotic-range proteinuria and malignant hypertension. Blood tests suggested the development of microangiopathic hemolytic anemia (MAHA) but the renal biopsy was mostly indicative of chronic changes associated with IgA nephropathy as well as vascular changes associated with malignant hypertension. Immunofluorescence exhibited deposits of IgA, C3, and IgM. Screening of the factor H gene revealed, in addition to the mutation, three heterozygous hemolytic uremic syndrome -associated risk polymorphisms (-257 c/t, 2089 a/g, and 2881 g/t) which may have increased the patient's susceptibility to the occurrence of MAHA triggered by malignant hypertension. The combined clinical picture of IgA nephropathy and MAHA may have been partly related to the alterations in factor H.

A novel heterozygous mutation in the clusterin gene, nucleotide position A1298C (glutamine > proline Q433P), was detected in exon 7 of a child with recurrent hemolytic uremic syndrome (HUS). The same mutation was found in the child's two siblings and mother but not in 120 controls. In addition, a previously described heterozygous mutation was detected in the gene encoding membrane cofactor protein (MCP) causing a 6 base-pair deletion 811-816delGACAGT in exon 6. It was found in the patient, both siblings and the father. One sibling had recovered from post-streptoccocal glomerulonephritis. Clusterin levels in the patient, siblings and parents were normal as was the migration pattern in a gel. Patient serum induced C3 and C9 deposition on normal washed platelets, and platelet activation, as detected by flow cytometry. The same phenomenon was found in serum taken from the siblings and the mother but not in the sample from the father and controls. Addition of clusterin to patient serum did not inhibit complement activation on platelets. The Q433P mutant, in isolated form, was further studied by binding to the components of the terminal complement complex. The mutant did not bind to C5b-7 that was immobilized onto a BIAcore chip, whereas wild-type clusterin did, indicating that the mutation could lead to defective inhibition of formation of the membrane attack complex under these conditions. Hemolysis of rabbit erythrocytes was inhibited by wild-type clusterin but not by the mutant. Mutated clusterin could thus not prevent assembly of the membrane attack complex on platelets and erythrocytes.

The kinin-forming pathway is activated on endothelial cells and neutrophils when high-molecular weight kininogen (HK) is cleaved by plasma kallikrein liberating bradykinin, a potent mediator of inflammation. Kinins are released during inflammatory conditions such as vasculitis, associated with neutrophil influx around blood vessels. Some patients with vasculitis have elevated plasma levels of neutrophil-derived proteinase 3 (PR3) and anti-PR3 Abs. This study investigated if neutrophil-derived PR3 could induce activation of the kinin pathway. PR3 incubated with HK, or a synthetic peptide derived from HK, induced breakdown and release of a novel tridecapeptide termed PR3-kinin, NH2-MKRPPGFSPFRSS-COOH, consisting of bradykinin with two additional amino acids on each terminus. The reaction was specific and inhibited by anti-PR3 and α1-antitrypsin. Recombinant wild-type PR3 incubated with HK induced HK breakdown, whereas mutated PR3, lacking enzymatic activity, did not. PR3-kinin bound to and activated human kinin B1 receptors, but did not bind to B2 receptors, expressed by transfected HEK293 cells in vitro. In human plasma PR3-kinin was further processed to the B2 receptor agonist bradykinin. PR3-kinin exerted a hypotensive effect in vivo through both B1 and B2 receptors as demonstrated using wild-type and B1 overexpressing rats as well as wild-type and B2 receptor knockout mice. Neutrophil extracts from vasculitis patients and healthy controls contained comparable amounts of PR3 and induced HK proteolysis, an effect that was abolished when PR3 was immunoadsorbed. Neutrophil-derived PR3 can proteolyze HK and liberate PR3-kinin, thereby initiating kallikrein-independent activation of the kinin pathway.

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathic disorder characterized by thrombocytopenia, hemolytic anemia, neurological and renal manifestations, and fever. It is associated with dysfunctional von Willebrand factor (VWF) proteolysis and the occurrence of VWF- and platelet-rich thrombi in the microcirculation of multiple organs, including the kidneys. Von Willebrand factor is a large glycoprotein that circulates in plasma as a series of multimers, and it plays a major role in primary hemostasis by inducing the formation of platelet plugs at sites of vascular injury and high-shear stress. Its activity is dependent on the sizes of the multimers, with ultra-large (UL) VWF multimers being biologically very potent. The ULVWF multimers are rapidly degraded upon their secretion from endothelial cells in normal individuals but not in the circulation of TTP patients, causing the formation of disseminated thrombi in the latter. The defective breakdown of VWF is attributed to a severely deficient activity of the VWF-cleaving protease ADAMTS13, a plasma metalloprotease synthesized in the liver, kidneys, and endothelium. This protease rapidly degrades VWF-platelet strings under flow by proteolytic cleavage of the VWF subunit, thereby regulating the size of the platelet thrombus. Congenital TTP occurs due to ADAMTS13 mutations, with the usual debut occurring during the first years of life, while acquired TTP is associated with auto-antibodies against ADAMTS13.