Ramesh Tati

Complement Activation Associated with ADAMTS13 Deficiency in Human and Murine Thrombotic Microangiopathy

This study addressed the contribution of ADAMTS13 deficiency to complement activation in thrombotic thrombocytopenic purpura (TTP). Renal tissue and blood samples were available from 12 TTP patients. C3 and C5b-9 deposition were demonstrated in the renal cortex of two TTP patients, by immunofluorescence and immunohistochemistry, respectively. C3 was also demonstrated in the glomeruli of Shiga toxin-2–treated Adamts13−/− mice (n = 6 of 7), but less in mice that were not Shiga toxin-2 treated (n = 1 of 8, p < 0.05) or wild-type mice (n = 0 of 7). TTP patient plasma (n = 9) contained significantly higher levels of complement-coated endothelial microparticles than control plasma (n = 13), as detected by flow cytometry. Exposure of histamine-stimulated primary glomerular endothelial cells to platelet-rich plasma from patients, or patient platelet-poor plasma combined with normal platelets, in a perfusion system, under shear, induced C3 deposition on von Willebrand factor–platelet strings (on both von Willebrand factor and platelets) and on endothelial cells. Complement activation occurred via the alternative pathway. No C3 was detected when cells were exposed to TTP plasma that was preincubated with EDTA or heat-inactivated, or to control plasma. In the perfusion system, patient plasma induced more release of C3- and C9-coated endothelial microparticles compared with control plasma. The results indicate that the microvascular process induced by ADAMTS13 deficiency triggers complement activation on platelets and the endothelium, which may contribute to formation of thrombotic microangiopathy.

Tissue Deposits of IgA-Binding Streptococcal M Proteins in IgA Nephropathy and Henoch-Schönlein Purpura

IgA nephropathy (IgAN) and Henoch-Schönlein purpura (HSP) are diseases characterized by IgA deposits in the kidney and/or skin. Both may arise after upper respiratory tract infections, but the pathogenic mechanisms governing these diseases remain unclear. Patients with IgAN (n = 16) and HSP (n = 17) were included in this study aimed at examining whether IgA-binding M proteins of group A streptococci could be involved. As M proteins vary in sequence, the study focused on the IgA-binding-region (IgA-BR) of three different M proteins: M4, M22, and M60. Renal tissue from IgAN and HSP patients and skin from HSP patients were examined for deposits of streptococcal IgA-BR by immunohistochemistry and electron microscopy using specific antibodies, and a skin sample from a HSP patient was examined by mass spectrometry. IgA-BR deposits were detected in 10/16 IgAN kidneys and 7/13 HSP kidneys. Electron microscopy demonstrated deposits of IgA-BRs in the mesangial matrix and glomerular basement membrane, which colocalized with IgA. Skin samples exhibited IgA-BR deposits in 4/5 biopsies, a result confirmed by mass spectrometry in one patient. IgA-BR deposits were not detected in normal kidney and skin samples. Taken together, these results demonstrate IgA-BR from streptococcal M proteins in patient tissues. IgA-BR, would on gaining access to the circulation, encounter circulatory IgA and form a complex with IgA-Fc that could deposit in tissues and contribute to the pathogenesis of IgAN and HSP.

Shiga toxin-mediated disease in MyD88-deficient mice infected with Escherichia coli O157:H7.

Toll-like receptors (TLRs) are key factors of innate immunity that detect pathogen invasion and trigger a host response. TLR4 can mediate a response through adaptor molecules, MyD88 or TRIF. In the present study, streptomycin-treated MyD88(-/-), Tlr4(-/-), Trif (Lps2/Lps2), and C57BL/6 wild-type (WT) mice were infected with either Shiga toxin (Stx)-producing or non-producing Escherichia coli O157:H7. Moderate to severe clinical signs of disease developed in MyD88(-/-) (n = 21/21), Tlr4(-/-) (n = 12/16), Trif (Lps2/Lps2) (n = 7/15) and WT mice (n = 6/20) infected with Stx-producing E. coli O157:H7 but not in mice inoculated with the Stx non-producing strain (n = 0/54, P < 0.001). MyD88(-/-) mice infected with Stx-producing E. coli O157:H7 developed the most severe disease and had the highest bacterial burden. Hematological analysis of sick MyD88(-/-) mice showed reduced red blood cell counts and reticulocytosis, suggesting hemolysis. Thrombocytopenia developed in MyD88(-/-), Trif (Lps2/Lps2), and WT mice, and creatinine levels were elevated in both MyD88(-/-) and WT mice infected with the Stx-producing strain. Renal histopathology showed evidence of glomerular capillary congestion, tubular desquamation, and fibrinogen deposition, and intestinal histopathology showed mucosal injury, edema, and inflammation in sick mice. Administration of purified Stx2 to MyD88(-/-) and WT mice led to severe disease in both groups, suggesting that MyD88(-/-) mice are not more sensitive to Stx than WT mice. As MyD88(-/-) mice developed the most severe disease hematological and pathological changes, the results suggest that dysfunctional innate immune responses via MyD88 enhanced Stx-induced disease.

Thrombotic microangiopathy mimicking membranoproliferative glomerulonephritis

A 4-year-old boy presented with proteinuria and developed progressive renal failure over 6 years. In the patients family, five individuals were affected with atypical haemolytic uraemic syndrome (aHUS) but not the patient. Renal biopsies (n = 3) showed glomerular basement membrane thickening with double contours, endothelial swelling and deposits of C3 and C1q. Electron microscopy revealed mesangial and subendothelial electron-dense deposits. Complement mutations in membrane cofactor protein (Y155D) and C3 (R713W and G1094R) were detected in all affected family members. The patient also had transient autoantibodies to factor H. The findings suggest that aHUS and glomerulopathy resembling membranoproliferative glomerulonephritis may have a common molecular background.

Biologically active ADAMTS13 is expressed in renal tubular epithelial cells.

ADAMTS13 mRNA, which encodes the von Willebrand factor-cleaving protease, has been detected in a variety of tissues, including the kidney. The aim of our study was to characterize tubular expression and bioactivity of ADAMTS13. ADAMTS13 mRNA was detected in cultured primary human renal tubular epithelial cells (HRTEC) and in A498 cells, a human renal carcinoma cell line, by real-time PCR. Protein was detected using immunofluorescence and immunoblotting. Immunoblots demonstrated that the protein was secreted. The protease was proteolytically active in both cell lysates and cleaved the FRETS–VWF73 substrate. ADAMTS13 was demonstrated in situ in the renal cortex by immunohistochemistry. Protease was detected in both the proximal and distal renal tubules in normal renal tissue (n = 3) as well as in patients with tubular disorders (n = 3). Immunoblotting revealed that ADAMTS13 was present in the urine of patients with tubulopathy (n = 5) but not in normal urine. ADAMTS13 in urine had a molecular size similar to that in plasma, which indicates that the protease originates in the tubuli because such large proteins do not normally pass the glomerular filter. In conclusion, human renal tubular epithelial cells synthesize biologically active ADAMTS13 which may, after release from tubuli, regulate hemostasis in the local microenvironment.

Haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) is defined by the simultaneous occurrence of nonimmune haemolytic anaemia, thrombocytopenia and acute renal failure. This leads to the pathological lesion termed thrombotic microangiopathy, which mainly affects the kidney, as well as other organs. HUS is associated with endothelial cell injury and platelet activation, although the underlying cause may differ. Most cases of HUS are associated with gastrointestinal infection with Shiga toxin‐producing enterohaemorrhagic Escherichia coli (EHEC) strains. Atypical HUS (aHUS) is associated with complement dysregulation due to mutations or autoantibodies. In this review, we will describe the causes of HUS. In addition, we will review the clinical, pathological, haematological and biochemical features, epidemiology and pathogenetic mechanisms as well as the biochemical, microbiological, immunological and genetic investigations leading to diagnosis. Understanding the underlying mechanisms of the different subtypes of HUS enables tailoring of appropriate treatment and management. To date, there is no specific treatment for EHEC‐associated HUS but patients benefit from supportive care, whereas patients with aHUS are effectively treated with anti‐C5 antibody to prevent recurrences, both before and after renal transplantation.

Complement Interactions with Blood Cells, Endothelial Cells and Microvesicles in Thrombotic and Inflammatory Conditions.

The complement system is activated in the vasculature during thrombotic and inflammatory conditions. Activation may be associated with chronic inflammation on the endothelial surface leading to complement deposition. Complement mutations allow uninhibited complement activation to occur on platelets, neutrophils, monocytes, and aggregates thereof, as well as on red blood cells and endothelial cells. Furthermore, complement activation on the cells leads to the shedding of cell derived-microvesicles that may express complement and tissue factor thus promoting inflammation and thrombosis. Complement deposition on red blood cells triggers hemolysis and the release of red blood cell-derived microvesicles that are prothrombotic. Microvesicles are small membrane vesicles ranging from 0.1 to 1 μm, shed by cells during activation, injury and/or apoptosis that express components of the parent cell. Microvesicles are released during inflammatory and vascular conditions. The repertoire of inflammatory markers on endothelial cell-derived microvesicles shed during inflammation is large and includes complement. These circulating microvesicles may reflect the ongoing inflammatory process but may also contribute to its propagation. This overview will describe complement activation on blood and endothelial cells and the release of microvesicles from these cells during hemolytic uremic syndrome, thrombotic thrombocytopenic purpura and vasculitis, clinical conditions associated with enhanced thrombosis and inflammation.

Complement activation in thrombotic microangiopathy

The endothelium lining the vascular lumen is continuously exposed to complement from the circulation. When erroneously activated on host cells, complement may generate a deleterious effect on the vascular wall leading to endothelial injury, exposure of the subendothelial matrix and platelet activation. In this review the contribution of complement activation to formation and maintenance of the pathological lesion termed thrombotic microangiopathy (TMA) is discussed. TMA is defined by vessel wall thickening affecting mainly arterioles and capillaries, detachment of the endothelial cell from the basement membrane and intraluminal thrombosis resulting in occlusion of the vessel lumen. The TMA lesion occurs in haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). HUS is further sub-classified as associated with Shiga toxin-producing Escherichia coli (STEC-HUS) or with complement dysregulation (atypical HUS) as well as other less common forms. The contribution of dysregulated complement activation to endothelial injury and platelet aggregation is reviewed as well as specific complement involvement in the development of HUS and TTP.

Phenotypic Expression of ADAMTS13 in Glomerular Endothelial Cells

Background ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13+/+) and deficient (Adamts13−/−) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells. Methodology/Principal Findings Immunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13+/+ mice but no staining was visible in tissue from Adamts13−/− mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13−/− mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF. Conclusions/Significance Glomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries.

C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles

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<0.001). Compared with microvesicles from wild-type cells, B1 receptor-positive microvesicles derived from transfected human embryonic kidney cells induced a significant neutrophil chemotactic effect, and a B1 receptor antagonist blocked this effect. Likewise, patient plasma induced neutrophil chemotaxis, an effect decreased by reduction of microvesicle levels and by blocking the B1 receptor. We used a perfusion system to study the effect of patient plasma (n=6) and control plasma (n=6) on the release of microvesicles from glomerular endothelial cells. Patient samples induced the release of significantly more B1 receptor-positive endothelial microvesicles than control samples, an effect abrogated by reduction of the microvesicles in the perfused samples. Perfusion of C1-inhibitor-depleted plasma over glomerular endothelial cells promoted excessive release of B1 receptor-positive endothelial microvesicles compared with normal plasma, an effect significantly decreased by addition of C1-inhibitor or B1 receptor-antagonist. Thus, B1 receptor-positive endothelial microvesicles may contribute to chronic inflammation by inducing neutrophil chemotaxis, and the reduction of these microvesicles by C1-inhibitor should be explored as a potential treatment for neutrophil-induced inflammation.