Systemic and local complement activation in peritoneal dialysis patients via conceivably distinct pathways.

BACKGROUND: Despite several advantages compared to haemodialysis (HD), peritoneal dialysis (PD) remains an underused dialysis technique due to its high technique failure rate related to membrane fibrosis and peritonitis events. Previous work has suggested a harmful role for the complement system in these processes, highlighting the need for a more comprehensive examination in PD. METHODS: Plasma levels of C1q, mannose-binding lectin (MBL), Properdin, Factor D, C3d/C3-ratio and soluble membrane attack complex (sC5b-9) were determined in PD patients (n = 55), HD patients (n = 41), non-dialysis chronic kidney disease (CKD) patients (n = 15) and healthy controls (n = 14). Additionally, C1q, MBL, Properdin, Factor D and sC5b-9 levels were assessed in the peritoneal dialysis fluid (PDF). In a subgroup, interleukin-6, matrix metalloproteinase-2 (MMP-2), myeloperoxidase (MPO) and elastase were measured in the PDF. RESULTS: PD patients had significantly higher systemic levels of sC5b-9 compared to healthy controls, CKD and HD patients (p < 0.001). Plasma levels of C1q and C3d/C3-ratios were significantly associated with systemic sC5b-9 levels (p < 0.001). Locally, sC5b-9 was detected in the PDF of all PD patients, and levels were approximately 33% of those in matched plasma, but they did not correlate. In the PDF, only Properdin levels remained significantly associated with PDF sC5b-9 levels in multivariate analysis (p < 0.001). Additionally, PDF levels of sC5b-9 positively correlated with elastase, MPO and MMP-2 levels in the PDF (p < 0.01). CONCLUSIONS: Our data reveal both systemic and local complement activation in PD patients. Furthermore, these two processes seem independent considering the involvement of different pathways and the lack of correlation.

Complement Is Activated During Normothermic Machine Perfusion of Porcine and Human Discarded Kidneys.

Background: The gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP. Methods: NMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition. Results: Complement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death. Conclusion: Normothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.

Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney.

Background: The role of the complement system in antibody-mediated rejection (ABMR) is insufficiently understood. We aimed to investigate the role of local and systemic complement activation in active (aABMR). We quantified complement activation markers, C3, C3d, and C5b-9 in plasma of aABMR, and acute T-cell mediated rejection (aTCMR), and non-rejection kidney transplant recipients. Intra-renal complement markers were analyzed as C4d, C3d, C5b-9, and CD59 deposition. We examined in vitro complement activation and CD59 expression on renal endothelial cells upon incubation with human leukocyte antigen antibodies. Methods: We included 50 kidney transplant recipients, who we histopathologically classified as aABMR (n=17), aTCMR (n=18), and non-rejection patients (n=15). Results: Complement activation in plasma did not differ across groups. C3d and C4d deposition were discriminative for aABMR diagnosis. Particularly, C3d deposition was stronger in glomerular (P<0,01), and peritubular capillaries (P<0,05) comparing aABMR to aTCMR rejection and non-rejection biopsies. In contrast to C3d, C5b-9 was only mildly expressed across all groups. For C5b-9, no significant difference between aABMR and non-rejection biopsies regarding peritubular and glomerular C5b-9 deposition was evident. We replicated these findings in vitro using renal endothelial cells and found complement pathway activation with C4d and C3d, but without terminal C5b-9 deposition. Complement regulator CD59 was variably present in biopsies and constitutively expressed on renal endothelial cells in vitro. Conclusion: Our results indicate that terminal complement might only play a minor role in late aABMR, possibly indicating the need to re-evaluate the applicability of terminal complement inhibitors as treatment for aABMR.

MASP-2 Is a Heparin-Binding Protease; Identification of Blocking Oligosaccharides.

It is well-known that heparin and other glycosaminoglycans (GAGs) inhibit complement activation. It is however not known whether fractionation and/or modification of GAGs might deliver pathway-specific inhibition of the complement system. Therefore, we evaluated a library of GAGs and their derivatives for their functional pathway specific complement inhibition, including the MASP-specific C4 deposition assay. Interaction of human MASP-2 with heparan sulfate/heparin was evaluated by surface plasmon resonance, ELISA and in renal tissue. In vitro pathway-specific complement assays showed that highly sulfated GAGs inhibited all three pathways of complement. Small heparin- and heparan sulfate-derived oligosaccharides were selective inhibitors of the lectin pathway (LP). These small oligosaccharides showed identical inhibition of the ficolin-3 mediated LP activation, failed to inhibit the binding of MBL to mannan, but inhibited C4 cleavage by MASPs. Hexa- and pentasulfated tetrasaccharides represent the smallest MASP inhibitors both in the functional LP assay as well in the MASP-mediated C4 assay. Surface plasmon resonance showed MASP-2 binding with heparin and heparan sulfate, revealing high Kon and Koff rates resulted in a Kd of ~2 µM and confirmed inhibition by heparin-derived tetrasaccharide. In renal tissue, MASP-2 partially colocalized with agrin and heparan sulfate, but not with activated C3, suggesting docking, storage, and potential inactivation of MASP-2 by heparan sulfate in basement membranes. Our data show that highly sulfated GAGs mediated inhibition of all three complement pathways, whereas short heparin- and heparan sulfate-derived oligosaccharides selectively blocked the lectin pathway via MASP-2 inhibition. Binding of MASP-2 to immobilized heparan sulfate/heparin and partial co-localization of agrin/heparan sulfate with MASP, but not C3b, might suggest that in vivo heparan sulfate proteoglycans act as a docking platform for MASP-2 and possibly prevent the lectin pathway from activation.

Strong predictive value of mannose-binding lectin levels for cardiovascular risk of hemodialysis patients.

BACKGROUND: Hemodialysis patients have higher rates of cardiovascular morbidity and mortality compared to the general population. Mannose-binding lectin (MBL) plays an important role in the development of cardiovascular disease. In addition, hemodialysis alters MBL concentration and functional activity. The present study determines the predictive value of MBL levels for future cardiac events (C-event), cardiovascular events (CV-event) and all-cause mortality in HD patients. METHODS: We conducted a prospective study of 107 patients on maintenance hemodialysis. Plasma MBL, properdin, C3d and sC5b-9 was measured before and after one dialysis session. The association with future C-events, CV-events, and all-cause mortality was evaluated using Cox regression models. RESULTS: During median follow-up of 27 months, 36 participants developed 21 C-events and 36 CV-events, whereas 37 patients died. The incidence of C-events and CV-events was significantly higher in patients with low MBL levels (<319 ng/mL, lower quartile). In fully adjusted models, low MBL level was independently associated with increased CV-events (hazard ratio 3.98; 95 % CI 1.88-8.24; P < 0.001) and C-events (hazard ratio 3.96; 95 % CI 1.49-10.54; P = 0.006). No association was found between low MBL levels and all-cause mortality. Furthermore, MBL substantially improved risk prediction for CV-events beyond currently used clinical markers. CONCLUSIONS: Low MBL levels are associated with a higher risk for future C-events and CV-events. Therefore, MBL levels may help to identify hemodialysis patients who are at risk to develop cardiovascular disease.

The antitumor activity of hydrophobin SC3, a fungal protein.

The use of mushroom extracts has been common practice in traditional medicine for centuries, including the treatment of cancer. Proteins called hydrophobins are very abundant in mushrooms. Here, it was examined whether they have antitumor activity. Hydrophobin SC3 of Schizophyllum commune was injected daily intraperitoneally starting 1 day after tumor induction in two tumor mouse models (sarcoma and melanoma). SC3 reduced the size and weight of the melanoma significantly, but the sarcoma seemed not affected. However, microscopic analysis of the tumors 12 days after induction revealed a strong antitumor effect of SC3 on both tumors. The mitotic activity of the tumor decreased 1.6- (melanoma) to 2.3-fold (sarcoma), while the vital mass decreased 2.3- (melanoma) to 4.3-fold (sarcoma) compared to the control. Treatment did not cause any signs of toxicity. Behavior, animal growth, and weight of organs were similar to animals injected with vehicle, and no histological abnormalities were found in the organs. In vitro cell culture studies revealed no direct cytotoxic effect of SC3 towards sarcoma cells, while cytotoxic activity was observed towards melanoma cells at a high SC3 concentration. Daily treatment with SC3 did not result in detectable levels of anti-SC3 antibodies in the plasma. Instead, a cellular immune response was observed. Incubation of spleen cells with SC3 resulted in a 1.5- to 2.5-fold increase in interleukin-10 and TNF-α mRNA levels. In conclusion, the nontoxic fungal hydrophobin SC3 showed tumor-suppressive activity possibly via immunomodulation and may be of benefit as adjuvant in combination with chemotherapy and radiation.

Oral and pulmonary delivery of thioether-bridged angiotensin-(1-7).

Instability and proteolytic degradation limit the delivery options and in vivo efficacy of many therapeutic peptides. We previously generated a thioether stabilized angiotensin-(1-7) analog, cAng-(1-7), which is resistant against proteolytic degradation in the circulation. We here investigated oral and pulmonary delivery of this compound. In a first step we investigated the in vitro stability of the peptide under conditions that mimic those that will be met after oral administration. We demonstrated that cAng-(1-7) is stable at pH 2.0, a pH value close to that of the stomach, has enhanced resistance to breakdown by proteases from pancreas at pH 7.4, and is resistant to breakdown by proteases from liver at the lysosomal pH 5.0. We subsequently demonstrated that, in the absence of any delivery system or formulation, cAng-(1-7) can be delivered orally and via the lung, with bioavailabilities of 0.28+/-0.05% and 28+/-5%, whereas drug uptake was maximal after subcutaneous administration (bioavailability of 98+/-6%). Therapeutic concentrations could be reached via all three routes of administration. The data prove that introduction of a thioether bridge in peptides opens novel delivery options for medically important peptides.

Use of hydrophobins in formulation of water insoluble drugs for oral administration.

The poor water solubility of many drugs requires a specific formulation to achieve a sufficient bioavailability after oral administration. Suspensions of small drug particles can be used to improve the bioavailability. We here show that the fungal hydrophobin SC3 can be used to make suspensions of water insoluble drugs. Bioavailability of two of these drugs, nifedipine and cyclosporine A (CyA), was tested when administered as a SC3-based suspension. SC3 (in a 1:2 (w/w) drug:SC3 ratio) or 100% PEG400 increased the bioavailability of nifedipine to a similar degree (6+/-2- and 4+/-3-fold, respectively) compared to nifedipine powder without additives. Moreover, SC3 (in a 7:1 (w/w) drug:hydrophobin ratio) was as effective as a 20-fold diluted Neoral formulation by increasing bioavailability of CyA 2.3+/-0.3-fold compared to CyA in water. Interestingly, using SC3 in the CyA formulation resulted in a slower uptake (p<0.001 in T(max)) of the drug, with a lower peak concentration (C(max) 1.8 mg ml(-1)) at a later time point (T(max) 9+/-2 h) compared to Neoral (C(max) 2.2 mg ml(-1); T(max) 3.2+/-0.2). Consequently, SC3 will result in a more constant, longer lasting drug level in the body. Taken together, hydrophobins are attractive candidates to formulate hydrophobic drugs.

Angiotensin-(1-7) with thioether bridge: an angiotensin-converting enzyme-resistant, potent angiotensin-(1-7) analog.

The in vivo efficacy of many therapeutic peptides is hampered by their rapid proteolytic degradation. Cyclization of these therapeutic peptides is an excellent way to render them more resistant against breakdown. Here, we describe the enzymatic introduction of a thioether ring in angiotensin [Ang-(1-7)], a heptapeptide that plays a pivotal role in the renin-angiotensin system and possesses important therapeutic activities. The lactic acid bacterium Lactococcus lactis, equipped with the plasmid-based nisin modification machinery, was used to produce thioether-bridged Ang-(1-7). The resulting cyclized Ang-(1-7) is fully resistant against purified angiotensin-converting enzyme, has significantly increased stability in homogenates of different organs and in plasma derived from pig, and displays a strongly (34-fold) enhanced survival in Sprague-Dawley (SD) rats in vivo. With respect to functional activity, cyclized Ang-(1-7) induces relaxation of precontracted SD rat aorta rings in vitro. The magnitude of this effect is 2-fold larger than that obtained for natural Ang-(1-7). The Ang-(1-7) receptor antagonist D-Pro(7)-Ang-(1-7), which completely inhibits the activity of natural Ang-(1-7), also abolishes the vasodilation by cyclized Ang-(1-7), providing evidence that cyclized Ang-(1-7) also interacts with the Ang-(1-7) receptor. Taken together, applying a highly innovative enzymatic peptide stabilization method, we generated a stable Ang-(1-7) analog with strongly enhanced therapeutic potential.

Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels.

The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1 e and is therefore only slightly cation-selective. Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known. We aimed to turn the essentially nonselective OmpF into a Ca2+-selective channel. To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117. Mutant OmpF containing this DEEE-locus has a high Ca2+ over Cl- selectivity and a Na+ current with a strongly increased sensitivity to 1 mM Ca2+. The charge/space competition model, initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects. By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca2+ channel. The results presented here fit quite well in the framework of charge/space competition theory.