Ultrastructural characterization of maturing iPSC-derived nephron structures upon transplantation.

Pluripotent stem cell-derived kidney organoids hold great promise as a potential auxiliary transplant tissue for individuals with end-stage renal disease and as a platform for studying kidney diseases and drug discovery. To establish accurate models, it is crucial to thoroughly characterize the morphological features and maturation stages of the cellular components within these organoids. Nephrons, the functional units of the kidney, possess distinct morphological structures that directly correlate with their specific functions. High spatial resolution imaging emerges as a powerful technique for capturing ultrastructural details that may go unnoticed with other methods such as immunofluorescent imaging and scRNA sequencing. In our study, we have applied software capable of seamlessly stitching virtual slides generated from electron microscopy, resulting in high-definition overviews of tissue slides. With this technology, we can comprehensively characterize the development and maturation of kidney organoids when transplanted under the renal capsule of mice. These organoids exhibit advanced ultrastructural developments upon transplantation, including the formation of the filtration barrier in the renal corpuscle, the presence of microvilli in the proximal tubule, and various types of cell sub-segmentation in the connecting tubule similarly to those seen in the adult kidney. Such ultrastructural characterization provides invaluable insights into the structural development and functional morphology of nephron segments within kidney organoids and how to advance them by interventions such as a transplantation. Research Highlights High-resolution imaging is crucial to determine morphological maturation of hiPSC-derived kidney organoids. Upon transplantation, refined ultrastructural development of nephron segments was observed, such as the development of the glomerular filtration barrier.

Characterization of the gene encoding component C3 of the complement system from the spider Loxosceles laeta venom glands: Phylogenetic implications.

A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3.

Loxosceles spider venom induces the release of thrombomodulin and endothelial protein C receptor: implications for the pathogenesis of intravascular coagulation as observed in loxoscelism.

BACKGROUND: The venom of the spider Loxosceles can cause both local and systemic effects including disseminated intravascular coagulation. AIM: The aim of this study was to investigate the effects of the venom of Loxosceles intermedia (L. intermedia) and the purified Sphingomyelinase D (SMaseD) toxin upon the Protein C (PC) natural anticoagulant pathway. RESULTS: Both the venom and e purified SMaseD reduced the cell surface expression of thrombomodulin (TM) and Endothelial PC Receptor on endothelial cells in culture. The reduction of cell surface expression was caused by cleavage from the cell surface mediated by activation of an endogenous metalloproteinase. Reduction of TM and Endothelial PC Receptor on the surface of these cells resulted in an impaired ability of the cells to assist in the thrombin-induced activation of PC. CONCLUSION: This novel observation gives further insight into the mechanisms of the pathology induced by venom from Loxosceles spiders and may aid the development of a suitable therapy.

Sphingomyelinases D induce direct association of C1q to the erythrocyte membrane causing complement mediated autologous haemolysis.

Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.

Variations in Loxosceles spider venom composition and toxicity contribute to the severity of envenomation.

Envenomation by Loxosceles spiders causes two main clinical manifestations: cutaneous and systemic loxoscelism. The factors contributing to the severity of loxoscelism are not fully understood. We have analysed biochemical and toxicity variations in venom of L. laeta and L. intermedia, with the aim to find a correlation with the seriousness of loxoscelism. Differences in expression of proteins, glycoproteins and sphingomyelinase activity were observed between venom from male and female spiders and between venom from the two species. These differences were reflected in the toxicity of the venoms including the capacity to induce complement-dependent haemolysis, dermonecrosis and lethality. Comparative analysis of gender and species, showed that these biological activities were more prominent in venom from female spiders, especially from L. laeta. Antiserum raised against venom from females L. laeta spiders had the highest efficacy in neutralizing venoms of males and females of both species. These results indicate that the severity of loxoscelism depends, at least partially, on the species and sex of the spider and suggest that for accidents involving L. laeta an specific serum therapy is necessary. Furthermore, it emphasizes the efficacy of the antiserum produced against L. laeta female venom in neutralizing Loxosceles venoms from different species and gender.

Role and regulation of pig CD59 and membrane cofactor protein/CD46 expressed on pig aortic endothelial cells.

BACKGROUND: Hyperacute rejection in xenotransplantation is caused by activation of complement (C) on endothelium. We have previously shown that purified C-regulators of the pig (CD59 and membrane cofactor protein [MCP]) are efficient regulators of human C (HuC). The aim of this study was to clarify the role of endogenously expressed C-regulatory molecules on pig endothelium in the protection against hyperacute rejection. METHODS: Porcine aortic endothelial cells (PAEC) were harvested and cultured for various passages. PAEC were examined for the expression of endogenous pig CD59 and MCP by flow cytometry. PAEC were assessed for their susceptibility to lysis by HuC. The effect of phorbol 12-myristate 13-acetate and various cytokines on the expression of MCP and CD59 and C-susceptibility was assessed. RESULTS: Primary PAEC showed an initial high level of expression of pig CD59, however, upon culturing, CD59 levels decreased dramatically to about 20% after five passages. In contrast, levels of MCP doubled upon culturing of PAEC to confluency and remained stable during at least five passages. Primary cells and cells in the early passages were more resistant to HuC than cells that were cultured for longer. Blocking the function of CD59 but not of MCP using monoclonal antibody increased the susceptibility to HuC. Purified human CD59 incorporated to a level of expression similar to that of pig CD59 reversed the increased C-susceptibility, suggesting that pig and human CD59 are similarly protective against HuC. Increase of C-resistance and of expression of pig MCP, but not of CD59, was achieved upon incubation with phorbol 12-myristate 13-acetate. Tumor necrosis factor-alpha, interleukin-1beta, interleukin-4, or interferon-gamma had no effect on C-regulator expression or C-susceptibility. CONCLUSIONS: These data demonstrate the importance of using primary PAEC or cells in the first passages of culturing in in vitro models of xenotransplantation and show that pig MCP and, in particular, pig CD59 play an important role in protection of PAEC from HuC.

Production and functional characterization of a soluble recombinant form of mouse CD59.

This report describes the engineering, expression, purification and functional characterization of a soluble recombinant form of murine CD59 (srMoCD59). We report the expression in Chinese hamster ovary (CHO) cells of a modified mouse CD59 cDNA that had been truncated at D-74, resulting in the loss of the glycosylphosphatidyl inositol (GPI) anchor, and containing six additional C-terminal histidines. The expressed srMoCD59 was purified from tissue culture supernatant by means of its poly-histidine tag using immobilized metal affinity chromatography. In comparison with CD59 on mouse erythrocytes, the srMoCD59 had a reduced molecular weight (18-20 000 as compared with 20-28 000 for GPI-anchored srMoCD59). The terminal complement inhibitory capacity of this soluble recombinant protein was assessed using two methods: a cobra venom factor (CVF)-triggered 'reactive-lysis' system and a C5b-7 site assay. In both assays, srMoCD59 inhibited lysis by the sera from all three species tested in the rank order mouse > rat >> human. The amount of srMoCD59 required to produce 50% inhibition of lysis in the C5b-7 site assay, using purified terminal components to develop lysis, was 10-fold less than that required in the same assay when EDTA serum was used as a source of C8 and C9, or in the CVF reactive lysis system. These data indicate that the presence of serum markedly interfered with the activity of srMoCD59 and have important implications for the use of recombinant soluble CD59 analogues as therapeutic agents in complement-mediated diseases.

Loxosceles intermedia spider envenomation induces activation of an endogenous metalloproteinase, resulting in cleavage of glycophorins from the erythrocyte surface and facilitating complement-mediated lysis.

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement (C)-dependent intravascular hemolysis. The authors studied the mechanism of induction of C-induced hemolysis. Purified Loxosceles toxins rendered human erythrocytes susceptible to lysis by human C but did not have an effect on the E-bound C-regulators DAF, CR1, or CD59. However, incubation with venom toxins caused cleavage of glycophorin from the erythrocyte (E) surface, facilitating C activation and hemolysis. The results suggest that glycophorin is an important factor in the protection of E against homologous C. Cleavage of glycophorin (GP) A, GPB, and GPC occurred at sites close to the membrane but could not be accomplished using purified GPA and purified toxins, demonstrating that cleavage was not an effect of a direct proteolytic action of the Loxosceles toxins on the glycophorins. Inhibition of the cleavage of glycophorins induced by Loxosceles venom was achieved with 1,10-phenanthroline. The authors propose that the sphingomyelinase activity of the toxins induces activation of an endogenous metalloproteinase, which then cleaves glycophorins. They observed the transfer of C-dependent hemolysis to other cells, suggesting that the Loxosceles toxins can act on multiple cells. This observation can explain the extent of hemolysis observed in patients after envenomation. Identification of the mechanism of induction of susceptibility to C-mediated lysis after Loxosceles envenomation opens up the possibility of the development of an effective therapeutic strategy. (Blood. 2000;95:683-691)

Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP).

Pig membrane cofactor protein (MCP; CD46) is a 50 000-60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals - a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I.

Characterization in vitro and in vivo of the pig analogue of human CD59 using new monoclonal antibodies.

CD59 is the sole characterized regulator of the complement membrane attack complex in humans. It is very widely and abundantly distributed, being present on all circulating cells, endothelia and epithelia, and in most tissues. CD59 analogues in rodents are distributed similarly. Interest in complement regulation in the pig has developed out of the current enthusiasm to exploit this species as a donor in xenotransplantation of organs to humans. We have recently isolated and cloned the pig analogue of human CD59. We here report the development and characterization of monoclonal antibodies against pig CD59. We have used these antibodies to develop efficient methods for the purification of pig CD59 to homogeneity from erythrocyte membranes and have obtained new information on the structure and function of the purified protein. The antibodies were found to function well in immunohistochemistry and have been used to perform a comprehensive survey of the expression and distribution of pig CD59 on cells and in organs of normal pigs. Pig CD59, like human CD59, is broadly expressed but there are some striking differences in tissue distribution, notably the apparent lack of pig CD59 on circulating platelets and on a subset of leucocytes in blood and lymphoid organs. The reported findings have important implications for the current approaches to avoiding complement-mediated hyperacute rejection in pig-to-human xenografts.

Sphingomyelinases in the venom of the spider Loxosceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.

Mechanisms of complement resistance induced by non-lethal complement attack and by growth arrest.

Non-lethal complement (C) attack on K562 cells has been shown to induce a transient resistance to lethal amounts of C. We have previously shown that incubation of K562 with phorbol 12-myristate 13-acetate (PMA) caused an increase in both CD59 expression and resistance to C killing and we were interested to examine whether non-lethal C attack caused a similar effect. We here demonstrate that expression of the C inhibitors decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59 was unaltered on K562 after non-lethal C attack and that neutralization of these inhibitors with specific blocking antibodies did not reverse the induced resistance. In an effort to understand the mechanisms of resistance we searched for other conditions that might induce C resistance in K562 cells. Growth-arrested cells showed a similar degree of resistance to C killing. The levels of DAF and MCP on these cells were unaltered whereas expression of CD59 was markedly reduced. Non-lethal C attack on these growth-arrested cells induced a further increase in resistance to C killing, suggesting that the mechanisms of resistance were not identical. Indeed, resistance of non-lethally attacked cells was completely lost within 8 hr of attack whereas resistance of growth-arrested cells was detectable for up to 48 hr after returning to cell cycle. These data demonstrate that C resistance induced by two distinct strategies is not mediated by the known membrane C inhibitors. Resistance may be a result of the expression of a novel inhibitor or due to metabolic depletion, a likely common consequence of non-lethal C attack and induction of growth arrest, implying that cells take an active role in C-mediated killing.

The glycosylation of the complement regulatory protein, human erythrocyte CD59.

Human erythrocyte CD59 contains N- and O-glycans and a glycosylphosphatidylinositol (GPI) anchor, all of which have been analyzed in this study. The anchor consists principally of the minimum core glycan sequence Manalpha1-2Manalpha1-6Manalpha1-4GlcN-linked to a phosphatidylinositol moiety with the structure sn-1-O-alkyl(C18:0 and C18:1)-2-O-acyl(C20:4)glycerol-3-phospho-1-(2-O-palmitoyl(C16:0))myo- inositol. This structure is essentially identical to that of human erythrocyte cholinesterase (Deeg, M. A., Humphrey, D. R., Yang, S. H. , Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573-18580). This first comparison of GPI anchors from different proteins expressed in the same tissue suggests that human reticulocytes produce only one type of anchor structure. The N- and O-glycans were sequenced using a novel approach involving digestion of the total glycan pool with multiple enzyme arrays. The N-glycan pool contained families of bi-antennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. The predominant O-glycans were NeuNAcalpha2-3Galbeta1-3GalNAc and Galbeta1-3[NeuNAcalpha2-3]GalNAc. Inspection of a molecular model of CD59, based on the NMR solution structure of the extracellular domain and the structural data from this study, suggested several roles for the glycans, including spacing and orienting CD59 on the cell surface and protecting the molecule from proteases. This work completes the initial structural analysis of CD59, providing the most complete view of any cell surface glycoprotein studied to date.

Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59.

We have previously described the isolation and cloning of the rat analogue of the human complement inhibitor CD59 (hCD59). Using the rat CD59 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indicated that they contained an open reading frame encoding a 124 amino acid protein. Comparisons with the known sequences of hCD59 and rCD59 suggested that the clones contained a full-length cDNA encoding the mouse analogue of CD59 (mCD59). The cDNA encoded a 81-bp 5'-flanking region, a 23 amino acid NH2-signal peptide, a 101 amino acid coding region including putative N-glycosylation sites and a glycosyl phosphatidylinositol (GPI) anchoring signal, and approximately 0.8 kb 3'-untranslated flanking region. Reverse transcriptase PCR revealed the presence of mCD59 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chromosome 2, a region that includes areas syntenous with the location of the human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59-negative human cell line conferred protection against lysis by complement from rodent, human, and several other species, confirming that mCD59 was the functional analogue of hCD59 and that function was not species restricted. The expressed protein was glycosyl phosphatidylinositol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs raised against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutralized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a molecular mass of 22 to 24 kDa.

Purification and characterization of the pig analogue of human membrane cofactor protein (CD46/MCP).

A panel of mAbs were raised against pig lymphocytes. Seven mAbs immunoprecipitated a 50- to 60-kDa membrane-bound protein. This protein, termed JM4C8-Ag, was expressed on a wide variety of cells, including all circulating cells and cells of fibroblast, epithelial, and endothelial origin. The JM4C8-Ag was transmembrane-anchored and glycosylated. One of the Abs was used in immunoaffinity chromatography to isolate JM4C8-Ag from erythrocyte membranes. N-terminal amino acid analysis through the first 28 residues showed a 43% homology with the human complement regulatory molecule membrane cofactor protein (MCP; CD46). The purified protein had cofactor activity for factor I-mediated cleavage of human and pig C3b, confirming its identity as the pig analogue of human MCP. The purified protein also strongly inhibited lysis of rabbit erythrocytes by human and pig complement after activation of the classical or alternative pathway. This is the first report of a nonprimate analogue of MCP. The presence of a resident MCP on pig cells capable of acting as a cofactor in the control of human complement activation has consequences for the use of pig organs in xenotransplantation.

Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent.

CD59, an 18-20-kD complement inhibitor anchored to the membrane via glycosyl phosphatidylinositol (GPI), can induce activation of T cells and neutrophils upon cross-linking with antibody. GPI-anchored molecules cocluster in high mol wt detergent-resistant complexes containing tyrosine kinases that are implicated in the signaling pathway. Exogenous, incorporated GPI-anchored molecules are initially unable to induce activation, presumably because they are not associated with kinases. Here we demonstrate that erythrocyte-derived CD59 incorporated in a CD59-negative cell line acquires signaling capacity in a time-dependent manner. Confocal microscopy revealed an initial diffuse distribution of CD59 that became clustered within 2 h to give a pattern similar to endogenous GPI-anchored molecules. Gel filtration of detergent-solubilized cells immediately after incorporation revealed that CD59 was mainly monomeric, but after 3 h incubation all was in high mol wt complexes and had become associated with protein kinases. Newly incorporated CD59 did not deliver a Ca2+ signal upon cross-linking, but at a time when it had become clustered and associated with kinase activity, cross-linking induced a large calcium transient, indicating that CD59 had incorporated in a specialized microenvironment that allowed it to function fully as a signal-transducing molecule.