35 research outputs found
Different Aspects of Classical Pathway Overactivation in Patients With C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis
The rare and heterogeneous kidney disorder C3 glomerulopathy (C3G) is characterized by dysregulation of the alternative pathway (AP) of the complement system. C3G is often associated with autoantibodies stabilizing the AP C3 convertase named C3 nephritic factors (C3NeF). The role of classical pathway (CP) convertase stabilization in C3G and related diseases such as immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) remains largely unknown. Here, we investigated the CP convertase activity in patients with C3G and IC-MPGN. Using a refined two-step hemolytic assay, we measured the stability of CP convertases directly in the serum of 52 patients and 17 healthy controls. In four patients, CP convertase activity was prolonged compared to healthy controls, i.e. the enzymatic complex was stabilized. In three patients (2 C3G, 1 IC-MPGN) the convertase stabilization was caused by immunoglobulins, indicating the presence of autoantibodies named C4 nephritic factors (C4NeFs). Importantly, the assay also enabled detection of non-immunoglobulin-mediated stabilization of the CP convertase in one patient with C3G. Prolonged CP convertase activity coincided with C3NeF activity in all patients and for up to 70 months of observation. Crucially, experiments with C3-depleted serum showed that C4NeFs stabilized the CP C3 convertase (C4bC2a), that does not contain C3NeF epitopes. All patients with prolonged CP convertase activity showed clear signs of complement activation, i.e. lowered C3 and C5 levels and elevated levels of C3d, C3bc, C3bBbP, and C5b-9. In conclusion, this work provides new insights into the diverse aspects and (non-)immunoglobulin nature of factors causing CP convertase overactivity in C3G/IC-MPGN.</p
Interactions of Shiga-like toxin with human peripheral blood monocytes
The cytotoxic effect of Shiga-like toxin (Stx; produced by certain Escherichia coli strains) plays a central role in typical hemolytic uremic syndrome (HUS). It damages the renal endothelium by inhibiting the cellular protein synthesis. Also, the monocyte has a specific receptor for Stx but is not sensitive for the cytotoxic effect. In this work, monocytes were studied as a potential transporter for Stx to the renal endothelium. Coincubation of isolated human monocytes loaded with Stx and target cells (vero cells and human umbilical vascular endothelial cells) were performed. Transfer was determined by measuring the protein synthesis of target cells and by flow cytometry. Furthermore, the effect of a temperature shift on loaded monocytes was investigated. Stx-loaded monocytes reduced the protein synthesis of target cells. After adding an antibody against Stx, incomplete recovery occurred. Also, adding only the supernatant of coincubation was followed by protein synthesis inhibition. Stx detached from its receptor on the monocyte after a change in temperature, and no release was detected without this temperature shift. Although the monocyte plays an important role in the pathogenesis of HUS, it has no role in the transfer of Stx
Transferrin mutations at the glycosylation site complicate diagnosis of congenital disorders of glycosylation type I
Congenital disorders of glycosylation (CDG) form a group of metabolic disorders caused by deficient glycosylation of proteins and/or lipids. Isoelectric focusing (IEF) of serum transferrin is the most common screening method to detect abnormalities of protein N-glycosylation. On the basis of the IEF profile, patients can be grouped into CDG type I or CDG type II. Several protein variants of transferrin are known that result in a shift in isoelectric point (pI). In some cases, these protein variants co-migrate with transferrin glycoforms, which complicates interpretation. In two patients with abnormal serum transferrin IEF profiles, neuraminidase digestion and subsequent IEF showed profiles suggestive of the diagnosis of CDG type I. Mass spectrometry of tryptic peptides of immunopurified transferrin, however, revealed a novel mutation at the N-glycan attachment site. In case 1, a peptide with mutation p.Asn630Thr in the 2nd glycosylation site was identified, resulting in an additional band at disialotransferrin position on IEF. After neuraminidase digestion, a single band was found at the asialotransferrin position, indistinguishable from CDG type I patients. In case 2, a peptide with mutation p.Asn432His was found. These results show the use of mass spectrometry of transferrin peptides in the diagnostic track of CDG type I
Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters
Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal physiology and pharmacology. We have established and characterized a conditionally immortalized PT epithelial cell line (ciPTEC) obtained by transfecting and subcloning cells exfoliated in the urine of a healthy volunteer. The PT origin of this line has been confirmed morphologically and by the expression of aminopeptidase N, zona occludens 1, aquaporin 1, dipeptidyl peptidase IV and multidrug resistance protein 4 together with alkaline phosphatase activity. ciPTEC assembles in a tight monolayer with limited diffusion of inulin-fluorescein-isothiocyanate. Concentration and time-dependent reabsorption of albumin via endocytosis has been demonstrated, together with sodium-dependent phosphate uptake. The expression and activity of apical efflux transporter p-glycoprotein and of baso-lateral influx transporter organic cation transporter 2 have been shown in ciPTEC. This established human ciPTEC expressing multiple endogenous organic ion transporters mimicking renal reabsorption and excretion represents a powerful tool for future in vitro transport studies in pharmacology and physiology
Uremic Toxins Inhibit Transport by Breast Cancer Resistance Protein and Multidrug Resistance Protein 4 at Clinically Relevant Concentrations
During chronic kidney disease (CKD), there is a progressive accumulation of toxic solutes due to inadequate renal clearance. Here, the interaction between uremic toxins and two important efflux pumps, viz. multidrug resistance protein 4 (MRP4) and breast cancer resistance protein (BCRP) was investigated. Membrane vesicles isolated from MRP4- or BCRP-overexpressing human embryonic kidney cells were used to study the impact of uremic toxins on substrate specific uptake. Furthermore, the concentrations of various uremic toxins were determined in plasma of CKD patients using high performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Our results show that hippuric acid, indoxyl sulfate and kynurenic acid inhibit MRP4-mediated [3H]-methotrexate ([3H]-MTX) uptake (calculated Ki values: 2.5 mM, 1 mM, 25 µM, respectively) and BCRP-mediated [3H]-estrone sulfate ([3H]-E1S) uptake (Ki values: 4 mM, 500 µM and 50 µM, respectively), whereas indole-3-acetic acid and phenylacetic acid reduce [3H]-MTX uptake by MRP4 only (Ki value: 2 mM and IC50 value: 7 mM, respectively). In contrast, p-cresol, p-toluenesulfonic acid, putrescine, oxalate and quinolinic acid did not alter transport mediated by MRP4 or BCRP. In addition, our results show that hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid and phenylacetic acid accumulate in plasma of end-stage CKD patients with mean concentrations of 160 µM, 4 µM, 129 µM, 1 µM and 18 µM, respectively. Moreover, calculated Ki values are below the maximal plasma concentrations of the tested toxins. In conclusion, this study shows that several uremic toxins inhibit active transport by MRP4 and BCRP at clinically relevant concentrations
Overactivity of alternative Pathway convertases in Patients With complement-Mediated renal Diseases
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Functional implications of mutations in the human renal outer medullary potassium channel (ROMK2) identified in Bartter syndrome
Shiga Toxin Selectively Upregulates Expression of Syndecan-4 and Adhesion Molecule ICAM-1 in Human Glomerular Microvascular Endothelium
Hemolytic uremic syndrome (HUS) is a severe renal disease that is often preceded by infection with Shiga toxin (Stx)-producing Escherichia coli (STEC). The exact mechanism of Stx-mediated inflammation on human glomerular microvascular endothelial cells (HGMVECs) during HUS is still not well understood. In this study, we investigated the effect of Stx1 on the gene expression of proteins involved in leucocyte-mediated and complement-mediated inflammation. Our results showed that Stx1 enhances the mRNA and protein expression of heparan sulfate proteoglycan (HSPG) syndecan-4 in HGMVECs pre-stimulated with tumor necrosis factor α (TNFα). CD44 was upregulated on mRNA but not on protein level; no effect on the mRNA expression of other tested HSPGs glypican-1 and betaglycan was observed. Furthermore, Stx1 upregulated the mRNA, cell surface expression, and supernatant levels of the intercellular adhesion molecule-1 (ICAM-1) in HGMVECs. Interestingly, no effect on the protein levels of alternative pathway (AP) components was observed, although C3 mRNA was upregulated. All observed effects were much stronger in HGMVECs than in human umbilical endothelial cells (HUVECs), a common model cell type used in endothelial studies. Our results provide new insights into the role of Stx1 in the pathogenesis of HUS. Possibilities to target the overexpression of syndecan-4 and ICAM-1 for STEC-HUS therapy should be investigated in future studies