Podocyte protease activated receptor 1 stimulation in mice produces focal segmental glomerulosclerosis mirroring human disease signaling events.

About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/-) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6.

Human Th17 cells produce a soluble mediator that increases podocyte motility via signaling pathways that mimic PAR-1 activation.

The specific pathogenesis of idiopathic nephrotic syndrome (NS) is poorly understood, and the role of immune mediators remains contentious. However, there is good evidence for the role of a circulating factor, and we recently postulated circulating proteases as candidate factors. Immunosuppressive therapy with glucocorticoids (GCs) and T cell inhibitors are widely used in the clinical treatment of NS. Given that T helper (CD4+) cells expressing IL-17A (so-called Th17 cells) have recently been reported to be resistant to GC treatment, and GC resistance remains a major challenge in the management of NS, we hypothesized that Th17 cells produce a circulating factor that is capable of signaling to the podocyte and inducing deleterious phenotypic changes. To test this, we generated human Th17 cells from healthy volunteers and added the supernatants from these T cell cultures to conditionally immortalized human podocytes in vitro. This demonstrated that podocytes treated with Th17 cell culture supernatant, as well as with patient disease plasma, showed significant stimulation of JNK and p38 MAPK pathways and an increase in motility, which was blocked using a JNK inhibitor. We have previously shown that nephrotic plasma elicits a podocyte response via protease-activated receptor-1 (PAR-1). Stimulation of PAR-1 in podocytes elicited the same signaling response as Th17 cell culture supernatant treatment. Equally, protease inhibitors with Th17 cell culture treatment blocked the signaling response. This was not replicated by the reagents added to Th17 cell cultures or by IL-17A. Hence, we conclude that an undefined soluble mediator produced by Th17 cells mimics the deleterious effect of PAR-1 activation in vitro. Given the association between pathogenic subsets of Th17 cells and GC resistance, these observations have potential therapeutic relevance for patients with NS.

Clinical Laboratory Investigations and Antivenom Administration after Malayan Pit Viper (Calloselasma rhodostoma) Envenoming: A Retrospective Study from Southernmost Thailand.

The Malayan pit viper (MPV: Calloselasma rhodostoma) is a medically important venomous snake causing numerous envenomations in Thailand. Administration of specific snake antivenom is the only effective treatment for MPV-envenomed patients. However, inappropriate administration or misuse of snake antivenom is problematic in some remote areas of tropical countries where the snakebite envenoming rate is notable. Currently, the indications for administration of MPV antivenom are focused mainly on hematological factors. These include 1) venous clotting time > 20 min, 2) unclotted 20-minute whole-blood clotting time, 3) international normalized ratio > 1.2, 4) platelet count < 50 × 103/µL, 5) systemic bleeding, and 6) impending compartment syndrome. We aimed to determine the association between laboratory data and antivenom administration in MPV-envenomed patients. A retrospective study of data from 2016 to 2021 in Narathiwat Province, the southernmost province in Thailand, was conducted. A total of 838 MPV-bitten patients were included in this study. Local effects and systemic effects were observed in 58.8% and 27.7% of patients, respectively. Coagulopathies, which range from abnormal blood clotting to systemic bleeding, represented the majority of systemic effects. Acute kidney injury developed in 2.5% of patients. In this study, 57.3% of patients were considered appropriate antivenom recipients. Interestingly, the present study revealed that local bleeding and mild to moderate thrombocytopenia became the independent factors for inappropriate use of MPV antivenom. Reeducation and supervision regarding the rational use of snake antivenom are needed to minimize the misuse of antivenom.

GlomSpheres as a 3D co-culture spheroid model of the kidney glomerulus for rapid drug-screening.

The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-ß1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model's usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models.

DI*A and DI*B Allele Frequencies Among Southern Thai Blood Donors.

Diego (DI) blood group genotyping is clinically important in Asian populations. Data of Diego blood type among southern Thais is still unknown. This study aimed to report DI*A and DI*B allele frequencies in southern Thai blood donors and to estimate potential risk of Dia incompatibility and alloimmunization in Thai populations. DNA samples obtained from 427 southern Thai blood donors were genotyped for DI*A and DI*B alleles by polymerase chain reaction with sequence-specific primer. DI*A and DI*B allele frequencies among southern Thais were 0.0047 and 0.9953. Their frequencies were similar to those among American Native, Italian, Filipino, Alaska Native/Aleut and Hawaiian/Pacific Islander populations; while, the frequencies significantly differed from central and northern Thai, Southeast Asian, Brazilian, Southern Brazilian, Brazilian Japanese descendants, Japanese, Han Chinese, Chinese, and Korean populations (P < 0.05). The Dia incompatibility among southern Thais (0.93%) was lower than among central Thais (3.49%), corresponding to a significantly lower probability of Dia alloimmunization (P < 0.05). This is the first report of DI*A and DI*B allele frequencies among southern Thais, which is beneficial for not only creating information for estimating risk of alloimmunization, but also providing antigen-negative red cell donors to prevent both alloimmunization and adverse transfusion reactions.

Proteomics analysis of siRNA-mediated silencing of Wilms' tumor 1 in the MDA-MB-468 breast cancer cell line.

The Wilms' tumor 1 (WT1) gene encodes a zinc finger which appears to be a transcriptional activator or repressor for many genes involved in cell differentiation, growth and apoptosis. In order to determine the relationship between WT1 and related proteins, WT1 was silenced with small interfering RNA (siRNA) and the protein expression pattern was analyzed by proteomics analysis including one-dimensional gel electrophoresis (1-DE) and LC-MS/MS mass spectrometry. The results revealed that 14 proteins were expressed in WT1-silenced cells (siRNA(WT1)) and 12 proteins were expressed in the WT1-expressing cells (siRNA(neg)), respectively. These proteins may be classified by their functions in apoptosis, cell signaling, protein folding, gene expression, redox-regulation, transport, structural and unknown functions. Mitogaligin, an apoptosis-related molecule, was identified when WT1 was silenced while the proteins related to the signaling pathway were detected in both siRNA(neg) and siRNA(WT1) but the type of proteins were different. For example, the IBtK protein and the SH2 domain-containing protein were present in siRNA(WT1) conditions, while the platelet-derived growth factor receptor α (PDGFRA) and Rho guanine nucleotide exchange factor 1 (Rho-GEF 1) were expressed in siRNA(neg). Of these, Rho-GEF was selected for validation by western blot analysis and demonstrated to be present only in the presence of WT1. In conclusion, WT1 is related to mitogaligin via EGFR and behaves as an anti-apoptotic molecule. Moreover, WT1 may be associated with PDGFRA and Rho-GEF 1 that activates proliferation in MDA-MB-468 cells.