The modulatory effects of large and small extracellular vesicles from normal human urine on calcium oxalate crystallization, growth, aggregation, adhesion on renal cells, and invasion through extracellular matrix: An in vitro study.

Urinary extracellular vesicles (uEVs) play important roles in physiologic condition and various renal/urological disorders. However, their roles in kidney stone disease remain unclear. This study aimed to examine modulatory effects of large and small uEVs derived from normal human urine on calcium oxalate (CaOx) crystals (the main component in kidney stones). After isolation, large uEVs, small uEVs and total urinary proteins (TUPs) with equal (protein equivalent) concentration were added into various crystal assays to compare with the control (without uEVs or TUPs). TUPs strongly inhibited CaOx crystallization, growth, aggregation and crystal-cell adhesion. Large uEVs had lesser degree of inhibition against crystallization, growth and crystal-cell adhesion, and comparable degree of aggregation inhibition compared with TUPs. Small uEVs had comparable inhibitory effects as of TUPs for all these crystal assays. However, TUPs and large uEVs slightly promoted CaOx invasion through extracellular matrix, whereas small uEVs did not affect this. Matching of the proteins reported in six uEVs datasets with those in the kidney stone modulator (StoneMod) database revealed that uEVs contained 18 known CaOx stone modulators (mainly inhibitors). These findings suggest that uEVs derived from normal human urine serve as CaOx stone inhibitors to prevent healthy individuals from kidney stone formation.

The upregulation of lamin A/C as a compensatory mechanism during tight junction disruption in renal tubular cells mediated by calcium oxalate crystals.

Calcium oxalate monohydrate (COM), the most important crystal causing kidney stone disease, upregulates lamin A/C but downregulates zonula occludens-1 (ZO-1) in renal tubular cells. While roles for F-actin and α-tubulin and their association with ZO-1 are known to regulate COM-mediated tight junction (TJ) disruption, roles of lamin A/C and its interplay with ZO-1 in COM kidney stone model remain unclear and are thus the objectives of this study. Lamin A/C was knocked down in MDCK cells by silencing RNA specific for LMNA (siLMNA). Both wild-type (WT) and siLMNA cells were treated with COM for 48-h compared with the untreated (control) cells. Western blotting and immunofluorescence staining revealed upregulated lamin A/C and downregulated ZO-1 in the COM-treated WT cells. siLMNA successfully reduced lamin A/C expression in both control and COM-treated cells. Nonetheless, siLMNA did not reverse the effect of COM on the decreases in ZO-1 and transepithelial resistance, but further reduced their levels in both control and COM-treated cells. Protein-protein interaction analysis demonstrated that two cytoskeletal proteins (actin and tubulin) served as the linkers to connect lamin A/C with ZO-1 and occludin (both of which are the TJ proteins). Altogether, these data implicate that lamin A/C and ZO-1 are indirectly associated to control TJ function, and ZO-1 expression is regulated by lamin A/C. Moreover, COM-induced upregulation of lamin A/C most likely serves as a compensatory mechanism to cope with the downregulation of ZO-1 during COM-mediated TJ disruption.

Epithelial-mesenchymal plasticity in kidney fibrosis.

Epithelial-mesenchymal transition (EMT) is an important biological process contributing to kidney fibrosis and chronic kidney disease. This process is characterized by decreased epithelial phenotypes/markers and increased mesenchymal phenotypes/markers. Tubular epithelial cells (TECs) are commonly susceptible to EMT by various stimuli, for example, transforming growth factor-ß (TGF-ß), cellular communication network factor 2, angiotensin-II, fibroblast growth factor-2, oncostatin M, matrix metalloproteinase-2, tissue plasminogen activator (t-PA), plasmin, interleukin-1ß, and reactive oxygen species. Similarly, glomerular podocytes can undergo EMT via these stimuli and by high glucose condition in diabetic kidney disease. EMT of TECs and podocytes leads to tubulointerstitial fibrosis and glomerulosclerosis, respectively. Signaling pathways involved in EMT-mediated kidney fibrosis are diverse and complex. TGF-ß1/Smad and Wnt/ß-catenin pathways are the major venues triggering EMT in TECs and podocytes. These two pathways thus serve as the major therapeutic targets against EMT-mediated kidney fibrosis. To date, a number of EMT inhibitors have been identified and characterized. As expected, the majority of these EMT inhibitors affect TGF-ß1/Smad and Wnt/ß-catenin pathways. In addition to kidney fibrosis, these EMT-targeted antifibrotic inhibitors are expected to be effective for treatment against fibrosis in other organs/tissues.

Tight junction and kidney stone disease.

Defects of tight junction (TJ) are involved in many diseases related to epithelial cell functions, including kidney stone disease (KSD), which is a common disease affecting humans for over a thousand years. This review provides brief overviews of KSD and TJ, and summarizes the knowledge on crystal-induced defects of TJ in renal tubular epithelial cells (RTECs) in KSD. Calcium oxalate (CaOx) crystals, particularly COM, disrupt TJ via p38 MAPK and ROS/Akt/p38 MAPK signaling pathways, filamentous actin (F-actin) reorganization and α-tubulin relocalization. Stabilizing p38 MAPK signaling, reactive oxygen species (ROS) production, F-actin and α-tubulin by using SB239063, N-acetyl-L-cysteine (NAC), phalloidin and docetaxel, respectively, successfully prevent the COM-induced TJ disruption and malfunction. Additionally, genetic disorders of renal TJ, including mutations and single nucleotide polymorphisms (SNPs) of CLDN2, CLDN10b, CLDN14, CLDN16 and CLDN19, also affect KSD. Finally, the role of TJ as a potential target for KSD therapeutics and prevention is also discussed.

Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property.

Calcineurin inhibitors (CNIs) are widely used in organ transplantation to suppress immunity and prevent allograft rejection. However, some transplant patients receiving CNIs have hypocitraturia, hyperoxaluria and kidney stone with unclear mechanism. We hypothesized that CNIs suppress activities of urinary calcineurin, which may serve as the stone inhibitor. This study aimed to investigate effects of calcineurin B (CNB) on calcium oxalate monohydrate (COM) stone formation. Sequence and structural analyses revealed that CNB contained four EF-hand (Ca2+-binding) domains, which are known to regulate Ca2+ homeostasis and likely to affect COM crystals. Various crystal assays revealed that CNB dramatically inhibited COM crystallization, crystal growth and crystal aggregation. At an equal amount, degrees of its inhibition against crystallization and crystal growth were slightly inferior to total urinary proteins (TUPs) from healthy subjects that are known to strongly inhibit COM stone formation. Surprisingly, its inhibitory effect against crystal aggregation was slightly superior to TUPs. While TUPs dramatically inhibited crystal-cell adhesion, CNB had no effect on this process. Ca2+-affinity assay revealed that CNB strongly bound Ca2+ at a comparable degree as of TUPs. These findings indicate that CNB serves as a novel inhibitor of COM crystallization, growth and aggregation via its high Ca2+-affinity property.

Theranostic roles of machine learning in clinical management of kidney stone disease.

Kidney stone disease (KSD) is a common illness caused by deposition of solid minerals formed inside the kidney. The disease prevalence varies, based on sociodemographic, lifestyle, dietary, genetic, gender, age, environmental and climatic factors, but has been continuously increasing worldwide. KSD is a highly recurrent disease, and the recurrence rate is about 11% within two years after the stone removal. Recently, machine learning has been widely used for KSD detection, stone type prediction, determination of appropriate treatment modality and prediction of therapeutic outcome. This review provides a brief overview of KSD and discusses how machine learning can be applied to diagnostics, therapeutics and prognostics in clinical management of KSD for better therapeutic outcome.

Alpha-tubulin relocalization is involved in calcium oxalate-induced tight junction disruption in renal epithelial cells.

Microtubule (MT) is associated with tight junction (TJ) structure and function. While calcium oxalate monohydrate (COM) commonly causes TJ disruption, its effects on MT remain unknown. This study thus addressed the involvement of a major MT protein, α-tubulin, in COM-induced TJ disruption. Protein-protein interactions analysis demonstrated that α-tubulin directly interacted with a TJ protein, zonula occludens-1 (ZO-1). MDCK renal cells were polarized and incubated with COM crystals for 48 h. Western blotting showed that COM reduced ZO-1, but not α-tubulin, level. Immunofluorescence staining revealed COM-induced relocalization of α-tubulin from apical membranes to cytoplasm and ZO-1 disruption at cell borders. COM also mediated progressive fall of epithelial barrier function, represented by transepithelial resistance (TER), which reached the lowest at 12-h till the end of crystal exposure. Pretreatment of the cells with docetaxel, the MT/tubulin stabilizer, completely prevented such α-tubulin relocalization, ZO-1 disruption/down-regulation, and TER reduction. These data indicate that α-tubulin relocalization is involved in COM-induced TJ disruption in renal epithelial cells.

Current update on theranostic roles of cyclophilin A in kidney diseases.

Cyclophilin A (CyPA) or peptidylprolyl isomerase A (PPIA), an immunophilin with peptidyl-prolyl cis/trans isomerase (PPIase) activity, is an abundant cellular protein widely expressed across various cell types and tissues, including the kidney. Expression of CyPA in the kidney is relatively higher in proximal tubular epithelial cells than others along the nephron. Alterations in expression and secretion of CyPA play important roles in physiological processes and pathophysiology of several diseases affecting the kidney. Herein, we provide a brief overview of CyPA structural biology and present the current update on its theranostic roles in various kidney diseases, including diabetic nephropathy, acute kidney injury, chronic kidney disease, renal fibrosis, and nephrotoxicity associated with organ transplantation. Notably, the diagnostic/prognostic role for urinary CyPA in several of these kidney diseases is promising. Finally, future perspectives on the CyPA research, especially targeting CyPA for therapeutics, are discussed. A comprehensive understanding of the theranostic roles of CyPA in kidney diseases is expected to provide novel insights into the design of new therapeutic interventions and prevention strategies.

Updating on Roles of HIV Intrinsic Factors: A Review of Their Antiviral Mechanisms and Emerging Functions.

BACKGROUND: Host restriction factors are cellular proteins that inhibit specific steps of the viral life cycle. Since the 1970s, several new factors have been identified, including human immunodeficiency virus-1 (HIV-1) replication restriction. Evidence accumulated in the last decade has substantially broadened our understanding of the molecular mechanisms utilized to abrogate the HIV-1 life cycle. SUMMARY: In this review, we focus on the interaction between host restriction factors participating in the early phase of HIV-1 infection, particularly CA-targeting proteins. Host factors involved in the late phase of the replication cycle, such as viral assembly and egress factors, are also described. Additionally, current reports on well-known antiviral intrinsic factors, as well as other viral restriction factors with their emerging roles, are included. CONCLUSION: A comprehensive understanding of the interactions between viruses and hosts is expected to provide insight into the design of novel HIV-1 therapeutic interventions.

Occupation of a thermoresistant-scaffold (αRep) at SP1-NC cleavage site disturbs the function of HIV-1 protease.

HIV-1 nucleocapsid (NC) becomes an attractive target for the development of novel anti-HIV-1 agents. Discovering of non-antibody scaffolds that disrupt the function of NC will be a potential aspect for disturbing viral maturation process. Correspondingly, we explored the specific binding site of the thermoresistant-scaffold protein, αRep9A8 which formerly demonstrated the inhibitory effect on HIV-1 replication. The portion of Gag, CA21-SP1-NC has been used as a template for designing nine overlapping peptides (P4-P12). The P9 peptide showed the strongest binding activity followed by P8 and P12 respectively. The amino acid sequences on those peptides resemble the N-terminal domain of the NC proximity to the SP1-NC initial cleavage site and across the conserved CCHC zinc finger 1 (ZF1) of NC. The interaction KD between αRep9A8 with its target was 224.9 ± 57.4 nM. Consequently, αRep9A8 demonstrated the interference of the HIV-1 protease function by hindering a protease cleavage site. The released NC product from CA21-SP1-NC was diminished. The present study provided an additional information of αRep9A8 function in interfering of viral maturation processes resulting in the decremental efficiency of viral infectivity.

Semi-quantification of HIV-1 protease inhibitor concentrations in clinical samples of HIV-infected patients using a gold nanoparticle-based immunochromatographic assay.

Individual drug concentration data can be a valuable tool for the clinical management of antiretroviral therapy (ART) for the treatment of HIV infection. High performance liquid chromatography (HPLC) based assays are currently the gold standard for drug measurement but its high cost and requirement of technical expertise limits its widespread use. Simpler user-friendly and inexpensive detection assays are needed. A novel immunochromatographic (IC) strip test to detect HIV-1 protease inhibitors (PIs) was fabricated by combining the proteolysis activity of HIV-protease (PR) and an immunochromatographic reaction. The PIs-IC strip cut-off to detect lopinavir (LPV) concentrations was set at 1,000 ng mL-1. We evaluated this novel PIs-IC strip for the semi-quantification of HIV PIs in plasma samples collected from healthy subjects and HIV-infected patients receiving antiretroviral treatment with and without LPV. LPV plasma drug levels were quantified by HPLC and evaluated (blinded to the HPLC results) using the PIs-IC strip. Results of plasma samples tested using the PIs-IC strip were available within 5 min. Using the PIs-IC strip test the accuracy, specificity and sensitivity were 97.8%, 97.1%, and 100%, respectively, compare to the gold-standard assay, to detect LPV in human plasma samples. This novel PIs-IC strip test could be used as a simple tool for the rapid monitoring of PIs levels in HIV-infected patients, although further clinical evaluation is needed.

Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patients.

Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.

Impairment of a membrane-targeting protein translated from a downstream gene of a "self-cleaving" T2A peptide conjunction.

The requirement for reliable bicistronic or multicistronic vectors in gene delivery systems is at the forefront of bio/biomedical technology. A method that provides an efficient co-expression of multiple heterologous proteins would be valuable for many applications, especially in medical science for treating various types of disease. In this study, we designed and constructed a bicistronic expression vector using a self-cleaving 2A peptide derived from a virus of the insect Thosea asigna (T2A). This exhibited the most efficient cleavage of the 2A sequence. Two versions of the T2A-based vector were constructed by switching the DNA sequences encoding the proteins of interest, the N-myristoylated protein and the nuclear-homing protein, upstream and downstream of the 2A linker, respectively. Our results showed that similar levels of mRNA expression were found and 100% of cleavage efficiency of T2A was observed. Nevertheless, we also reported the cleared evidence that the N-myristoylated protein cannot be placed downstream of the 2A sequence. Since the protein product fails to translocate to the plasma membrane due to altered myristoylation process, the gene position of the T2A-based vector is meaningful for the subcellular localization of the N-myristoylated protein. Therefore, the observation was marked as a precaution for using the 2A peptide. To adopt the 2A peptide technology for generating the bicistronic or multicistronic expression, the vector design should be carefully considered for the transgene position, signal sequences, and post-translational modifications of each individual protein.

Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation.

A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (αRep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of αRep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions. Two strong αRep binders were isolated from the screen, αRep4E3 (32 kDa; 7 internal repeats) and αRep9A8 (28 kDa; 6 internal repeats). Their antiviral activity against HIV-1 was evaluated in VLP-producer cells and in human SupT1 cells challenged with HIV-1. Both αRep4E3 and αRep9A8 showed a modest but significant antiviral effects in all bioassays and cell systems tested. They did not prevent the proviral integration reaction, but negatively interfered with late steps of the HIV-1 life cycle: αRep4E3 blocked the viral genome packaging, whereas αRep9A8 altered both virus maturation and genome packaging. Interestingly, SupT1 cells stably expressing αRep9A8 acquired long-term resistance to HIV-1, implying that αRep proteins can act as antiviral restriction-like factors.

Combined Antiviral Therapy Using Designed Molecular Scaffolds Targeting Two Distinct Viral Functions, HIV-1 Genome Integration and Capsid Assembly.

Designed molecular scaffolds have been proposed as alternative therapeutic agents against HIV-1. The ankyrin repeat protein (Ank(GAG)1D4) and the zinc finger protein (2LTRZFP) have recently been characterized as intracellular antivirals, but these molecules, used individually, do not completely block HIV-1 replication and propagation. The capsid-binder Ank(GAG)1D4, which inhibits HIV-1 assembly, does not prevent the genome integration of newly incoming viruses. 2LTRZFP, designed to target the 2-LTR-circle junction of HIV-1 cDNA and block HIV-1 integration, would have no antiviral effect on HIV-1-infected cells. However, simultaneous expression of these two molecules should combine the advantage of preventive and curative treatments. To test this hypothesis, the genes encoding the N-myristoylated Myr(+)Ank(GAG)1D4 protein and the 2LTRZFP were introduced into human T-cells, using a third-generation lentiviral vector. SupT1 cells stably expressing 2LTRZFP alone or with Myr(+)Ank(GAG)1D4 showed a complete resistance to HIV-1 in viral challenge. Administration of the Myr(+)Ank(GAG)1D4 vector to HIV-1-preinfected SupT1 cells resulted in a significant antiviral effect. Resistance to viral infection was also observed in primary human CD4+ T-cells stably expressing Myr(+)Ank(GAG)1D4, and challenged with HIV-1, SIVmac, or SHIV. Our data suggest that our two anti-HIV-1 molecular scaffold prototypes are promising antiviral agents for anti-HIV-1 gene therapy.

A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.

Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.