Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. Complement activation split products C3c, C3dg and sC5b-9 associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEV) were enriched by lectin- and immunoaffinity-isolation and analyzed by immunoblotting. Urine complement excretion increased significantly in KTRs with albumin/creatinine ratio ≥ 300 mg/g compared to < 30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 months after transplantation. The fractional excretion of C9 neoantigen was significantly higher than for albumin indicating post-filtration generation. C9 neoantigen was detected in uEVs in six of nine of albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen positive KTRs, lectin-affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen and SGLT2 but only weakly for AQP2. Co-isolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9 associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.

Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

Target identification by phosphoproteomics: RIN1 modulation of sorafenib-induced cytotoxicity in renal cell carcinoma

e14543 Background: Sorafenib (SFB) is a multi-tyrosine kinase inhibitor clinically useful in treatment of metastatic renal cancer. While the inhibition of angiogenesis is considered a major mechanism of action, identification of targets regulating growth inhibitory effects of SFB is necessary to further improve its efficacy and reduce toxicity. Methods: In this study we used targeted phosphoproteomics to identify tyrosine phosphorylated proteins that are differentially affected in control and SFB-treated human CAKI-1 renal cell carcinoma cells. The strategy involved immunoaffinity isolation of phosphotyrosine containing proteins and liquid chromatography - tandem mass spectrometry (MS) for identification of candidate proteins. Results: Among identified proteins, signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were found to be hypophosphorylated in SFB-treated compared to untreated CAKI-1 cells based on quantitative MS analysis, by peptide counts and native peptide reference method. A ∼4-fold decrease in expression and phosphorylation of STAT1 was observed in cells treated with 10 μM SFB for 48h. Up to 8-fold SFB dose-dependent (5–15 μM) decrease in phosphorylation of RIN1 at tyrosine 36, but not in total RIN1 expression, was observed. Similar effects on decreased phosphorylation of STAT1 and RIN1 were also observed in 786-O renal cell carcinoma treated with SFB. Hypophosphorylation of RIN1 at tyrosine 36 was observed in CAKI-1 cells treated with 5 μM sunitinib but not with imatinib (≤ 10 μM). Treatment of CAKI-1 cells with RIN1 targeted, but not control si-RNA led to down-regulation of RIN1 expression and attenuation of antiproliferative effects of SFB. Notably, ∼2-fold higher expression of RIN1 protein (total and phosphorylated) was observed in CAKI-1 cells selected for resistance following continuous exposure to 7.5 μM SFB. However, unlike parent CAKI-1 cells, prolonged exposure of these SFB-resistant CAKI-1 cells to 7.5 μM SFB did not completely abrogate phosphorylation of RIN1 at tyrosine 36. Conclusions: These results demonstrate that RIN1, a Ras effector protein with multiple biochemical functions, is a target for the anti-tumor effects of SFB in kidney cancer cells. [Table: see text]

Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition

The failure of most non-ionic detergents to release patches of DRM (detergent-resistant membrane) at 37 °C undermines the claim that DRMs consist of lipid nanodomains that exist in an Lo (liquid ordered) phase on the living cell surface. In the present study, we have shown that inclusion of cations (Mg2+, K+) to mimic the intracellular environment stabilizes membranes during solubilization sufficiently to allow the isolation of DRMs at 37 °C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 h) stability at 37 °C, and are vesicles or sheets up to 150–200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1, but not PrP, DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37 °C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.

Quantification of Thyroglobulin, a Low-Abundance Serum Protein, by Immunoaffinity Peptide Enrichment and Tandem Mass Spectrometry

Background: Quantification of serum tumor markers plays an important role in determining whether patients treated for cancer require further therapy. Whereas large-scale proteomic efforts aim to identify novel tumor markers to facilitate early detection, optimization of methods for quantifying known tumor markers offers another approach to improving management of malignancies. For example, immunoassays used in clinical practice to measure established tumor markers suffer from potential interference from endogenous immunoglobulins and imperfect concordance across platforms—problems that also plague many other immunoassays. To address these important limitations, this study used peptide immunoaffinity enrichment in concert with liquid chromatography–tandem mass spectrometry (LC-MS/MS) to quantify thyroglobulin, a well-characterized tumor marker. Methods: We identified 3 peptides in tryptic digests of thyroglobulin that were detected at low concentrations by tandem mass spectrometry, raised polyclonal antibodies to those peptides, and used the antibodies to extract the 3 corresponding peptides from tryptic digests of human serum. We quantified each endogenous peptide using LC-MS/MS and multiple reaction monitoring with external calibrators. Results: The detection limit for endogenous thyroglobulin in serum was 2.6 μg/L (4 pmol/L). Direct comparison with immunoassay revealed good correlation (r2 = 0.81). Conclusions: Immunoaffinity peptide enrichment–tandem mass spectrometry can detect tryptic peptides of thyroglobulin at picomolar concentrations while also digesting the endogenous immunoglobulins that can potentially interfere with traditional immunoassays. Our observations suggest a general analytical strategy for using immunoaffinity isolation together with tandem mass spectrometry to quantify tumor antigens and other low-abundance proteins in human serum.

Evidence for a Superoxide Permeability Pathway in Endosomal Membranes

ABSTRACT The compartmentalized production of superoxide (·O2 −) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-κB following interleukin 1β (IL-1β) stimulation. It remains unclear how ·O2 − produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated ·O2 − movement out of IL-1β-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-κB activation. The relative outward permeability of NADPH-dependent ·O2 − from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for ·O2 −. In these studies, ∼60% of ·O2 − efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that ·O2 − and Cl− are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1β-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-κB following IL-1β stimulation. Together, these results suggest that ·O2 − exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of ·O2 − at the endosomal surface may produce the localized H2O2 required for redox-activation of NF-κB.

Epitope Extraction Technique Using a Proteolytic Magnetic Reactor Combined with Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry as a Tool for the Screening of Potential Vaccine Lead Peptides

Epitope extraction technique is based on the specific digestion of a target protein followed by immunoaffinity isolation of a specific recognition peptide. This technique, in combination with mass spectrometry, has been efficiently used for epitope identification. The major goal of this work was to utilize newly developed enzyme and immunoaffinity magnetic reactors for the epitope extraction procedure and confirm the efficiency of this improved system for epitope screening of proteins. Alginic acid-coated magnetite microparticles with immobilized TPCK-trypsin provided high working efficiency with low non-specific adsorption, digestion time in minutes and low frequency of missed cleavages. The sensitivity and specificity of tryptic fragmentation of the ß-amyloid-peptide Aβ (1–40) as a model polypeptide was confirmed by Fourier-transform ion cyclotron resonance mass spectrometry analysis. The Sepharose reactor or immunoaffinity magnetic reactors, both with anti-amyloid-β monoclonal antibodies, were used for specific isolation and identification of target peptides. In this way, the epitope extraction technique combined with mass spectrometric analysis is shown to be an excellent base for molecular screening of potential vaccine lead proteins.