Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

Beyond 2D: A scalable and highly sensitive method for a comprehensive 3D analysis of kidney biopsy tissue.

The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-µm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Brown Tumour in Chronic Kidney Disease: Revisiting an Old Disease with a New Perspective.

Osteitis fibrosa cystica (OFC) and Brown Tumours are two related but distinct types of bone lesions that result from the overactivity of osteoclasts and are most often associated with chronic kidney disease (CKD). Despite their potential consequences, these conditions are poorly understood because of their rare prevalence and variability in their clinical manifestation. Canonically, OFC and Brown Tumours are caused by secondary hyperparathyroidism in CKD. Recent literature showed that multiple factors, such as hyperactivation of the renin-angiotensin-aldosterone system and chronic inflammation, may also contribute to the occurrence of these diseases through osteoclast activation. Moreover, hotspot KRAS mutations were identified in these lesions, placing them in the spectrum of RAS-MAPK-driven neoplasms, which were until recently thought to be reactive lesions. Some risk factors contributed to the occurrence of OFC and Brown Tumours, such as age, gender, comorbidities, and certain medications. The diagnosis of OFC and Brown Tumours includes clinical symptoms involving chronic bone pain and laboratory findings of hyperparathyroidism. In radiological imaging, the X-ray and Computed tomography (CT) scan could show lytic or multi-lobular cystic alterations. Histologically, both lesions are characterized by clustered osteoclasts in a fibrotic hemorrhagic background. Based on the latest understanding of the mechanism of OFC, this review elaborates on the manifestation, diagnosis, and available therapies that can be leveraged to prevent the occurrence of OFC and Brown Tumours.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

MAGI-2 orchestrates the localization of backbone proteins in the slit diaphragm of podocytes.

Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. The slit diaphragm between the foot processes of podocytes functions as a final filtration barrier to prevent serum protein leakage into urine. The slit-diaphragm consists mainly of Nephrin and Neph1, and localization of these backbone proteins is essential to maintaining the integrity of the glomerular filtration barrier. However, the mechanisms that regulate the localization of these backbone proteins have remained elusive. Here, we focused on the role of membrane-associated guanylate kinase inverted 2 (MAGI-2) in order to investigate mechanisms that orchestrate localization of slit-diaphragm backbone proteins. MAGI-2 downregulation coincided with a reduced expression of slit-diaphragm backbone proteins in human kidneys glomerular disease such as focal segmental glomerulosclerosis or IgA nephropathy. Podocyte-specific deficiency of MAGI-2 in mice abrogated localization of Nephrin and Neph1 independently of other scaffold proteins. Although a deficiency of zonula occuldens-1 downregulated the endogenous Neph1 expression, MAGI-2 recovered Neph1 expression at the cellular edge in cultured podocytes. Additionally, overexpression of MAGI-2 preserved Nephrin localization to intercellular junctions. Co-immunoprecipitation and pull-down assays also revealed the importance of the PDZ domains of MAGI-2 for the interaction between MAGI-2 and slit diaphragm backbone proteins in podocytes. Thus, localization and stabilization of Nephrin and Neph1 in intercellular junctions is regulated mainly via the PDZ domains of MAGI-2 together with other slit-diaphragm scaffold proteins. Hence, these findings may elucidate a mechanism by which the backbone proteins are maintained.

A rare case of nephrotic syndrome associated with Dent's disease: a case report.

Dent's disease is a rare X-linked condition caused by a mutation in CLCN5 and OCRL gene, which impair the megalin-cubilin receptor-mediated endocytosis in kidney's proximal tubules. Thus, it may manifest as nephrotic-range low-molecular-weight proteinuria (LMWP). On the other hand, glomerular proteinuria, hypoalbuminemia, and edema formation are the key features of nephrotic syndrome that rarely found in Dent's disease. Here, we reported a man in his 30 s with Dent's disease presented with leg edema for 5 days. The laboratory results revealed hypoalbuminemia and a decrease of urine ß2-microglobulin/urine protein ratio (Uß2-MG /UP), indicating that the primary origin of proteinuria shifted from LMWP to glomerular proteins. The kidney biopsy revealed glomerular abnormality and calcium deposition in the renal medulla. Electron microscopy of the kidney tissue indicated extensive foot-process effacement of the glomerular podocytes and degeneration of tubular epithelium. After a combination of treatment with prednisolone and cyclosporine (CyA), the nephrotic syndrome was remitted. Given the atypical clinical presentation and the shift of LMWP to glomerular proteinuria in this patient, glomerulopathy and the Dent's disease existed separately in this patient.

MAGI-2 and scaffold proteins in glomerulopathy.

In many cells and tissues, including the glomerular filtration barrier, scaffold proteins are critical in optimizing signal transduction by enhancing structural stability and functionality of their ligands. Recently, mutations in scaffold protein membrane-associated guanylate kinase inverted 2 (MAGI-2) encoding gene were identified among the etiology of steroid-resistant nephrotic syndrome. MAGI-2 interacts with core proteins of multiple pathways, such as transforming growth factor-ß signaling, planar cell polarity pathway, and Wnt/ß-catenin signaling in podocyte and slit diaphragm. Through the interaction with its ligand, MAGI-2 modulates the regulation of apoptosis, cytoskeletal reorganization, and glomerular development. This review aims to summarize recent findings on the role of MAGI-2 and some other scaffold proteins, such as nephrin and synaptopodin, in the underlying mechanisms of glomerulopathy.

Distribution of dimethylarginine-dimethylaminohydrolase-II (DDAH2) gene polymorphism in hemodialysis patients.

AIM: to describe the distribution of single nucleotide polymorphisms (SNPs) at -449 (promoter region) DDAH 2 in hemodialysis (HD) patients. METHODS: this study was a descriptive study, 56 HD patients and 30 healthy individuals were enrolled. Based on its etiology, the HD patient group was further divided into hypertension (HT) group and non-HT group. DNA was extracted from whole blood samples with a commercially available DNA isolation kit. Genotyping of the polymorphisms was performed using PCR-based SNP detection methods (Applied Biosystems, Carlsbad, USA) based on 5'-exonuclease activity assays for rs805305 (-449 G/C). Allelic variation was assessed in each participant. RESULTS: heterozygotes were observed as the most abundant genotypes in both groups (70% in healthy individuals and 55% in HD patients), followed by GG genotype in the HD patients (30%), while CC (27%) was the second most common genotype polymorphism in the healthy individuals. CONCLUSION: there is a significant difference in distribution of DDAH 2 gene polymorphism among HD patients compared to healthy individuals (p=0.01).