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-β1, 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.

Aqueous fruit pulp extract of Adansonia digitata (L) protects against lead-acetate-induced hepato-renal damage in rat model

Background Adansonia digitata (L) fruit has a multi-purpose function one among many, is the antioxidant activities of the fruit by preventing oxidative stress. The effect of Adansonia digitata (L) fruit on lead-induced liver and kidney damage is not clear. Hence, the study was aimed to assessed the protective role of Adansonia digitata (L) fruits against lead acetate induced changes in the liver and kidney function test parameters and the histology of both organ in experimental rats. The rats were divided into five groups with five rats each. All the rats were administered with respective assigned treatment once daily for 6 weeks. Rats in groups I were administered with just distil water (2 ml/kg). Rats in groups II were administered with lead acetate (30 mg/kg) while rats in groups III–V were administered Adansonia digitatata (L) fruit extract (250 mg/kg and 500 mg/kg) and Succimer (5 mg/kg) respectively, then additionally challenged with lead acetate (30 mg/kg) immediately after. At the end of the administration, the blood serum from the experimental rats were used for biochemical analysis. Then, the the organs such as the liver and kidney collected for histological study. Results Rats administered with Lead acetate showed an increase in AST, ALP and ALT as well as increase in urea and creatinine level (p < 0.001), when compared with the control group (group I), where as Adansonia digitatata (L) fruit prevented the effect (upsurge of serum, Urea, Creatinine, AST, ALP and ALT) of lead acetate. Rats administer with only Lead acetate revealed marked liver steatosis and the degeneration of the kidney glomerulus. The Adansonia digitatata (L) fruit extract and Succimer prevented the histological liver steatosis, as well as the degeneration of the glomerulus of the kidney cytoarchitecture. Conclusion The findings in this study suggest that Adansonia digitata fruits extract has a protective potentials against lead acetate induced liver and kidney toxicity by preventing the upsurge of liver function enzymes and kidney function parameters. Hence, Adansonia digitata fruits can serve as a natural plant agent that can prevent hepato-renal toxicity. Therefore, Adansonia digitata holds future prospects in preclinical framework to ameliorate organs toxicity for oral therapeutic applications.

Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, therapeutics that can help manage the disease are still required until immunity has been achieved globally. The identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2-ΔOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in-line with reported proteinuria and liver damage in patients with COVID-19. Using the nano-luciferase as a measure of virus replication we identified 35 drugs that reduced replication in Vero cells and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

STUDY OF ACUTE AND SUB-ACUTE TOXICITY OF Boehmeria virgata (FORST) GUILL LEAF EXTRACT IN WISTAR RATS

This study was carried out to evaluate the acute and sub-acute toxicity of the standardized Boehmeria virgata leaf extract (BLVE) in Wistar rats. B. virgata is a traditional herb utilized by the people of Makassar, Indonesia to cure inflammation and cancer. In the current study, thirty (30) Wistar rats were divided into 6 groups (3 groups of males and 3 groups of females) for acute treatments. Similarly, for investigating sub-acute treatment forty (40) Wistar rats were split into 8 groups (4 groups of males and 4 groups of females). For acute toxicity treatment, selected rats received 2000 mg and 5000 mg/kg of BVLE by oral administration for 14 days while for the estimation of sub-acute toxicity, experimental rats were treated with 250, 500, and 1000 mg/kg BVLE for 28 days. After 14 days of treatment, the rats were monitored for any form of changes in behavior, weight, food, water intake, and histopathology. The treated animals underwent hematological, biochemical, histopathological, and organ weight analysis after 28 days. Results of the study revealed no significant differences in body and organ weight, intake of food, and water after acute BVLE treatment in rats compared with those in the control group. The histopathological study suggested a general hydropic degeneration of the liver after acute treatment with 5000 mg/kg; such degeneration did not occur in the kidneys and kidney glomerulus of BVLE treated rats. Further, no significant toxicity was shown in hematological, biochemical, organ weight, and histopathological data in the sub-acute BVLE group after comparing treated rats with the control group. Results of the study can be concluded that BVLE is not toxic at doses of up to 1000 mg/kg. Therefore B. virgate fulfilled a preclinical criterion that is necessary for its further establishment as a clinically useful extract.

Drosophila Filamin exhibits a mechano-protective role during nephrocyte injury via induction of hypertrophic growth

Podocytes are highly specialized epithelial cells of the kidney glomerulus and are an essential part of the filtration barrier. Due to their position and function in the kidney, they are exposed to constant biomechanical forces such as shear stress and hydrostatic pressure. These forces increase during disease, resulting in podocyte injury and loss. The mechanism by which biomechanical forces are sensed and transduced to elicit an adaptive and protective response remains largely unknown. Here we show, using the Drosophila nephrocyte model, that the mechanosensor Cheerio (dFilamin) is central to this mechano-protective mechanism. We found expression of an activated mechanosensitive variant of Cheerio induced hypertrophy and rescued filtration function in injured nephrocytes. Additional analysis with human Filamin B confirmed this mechano-protective role. We delineated the mechano-protective pathway downstream of Cheerio and found activation of TOR and Yorkie induce nephrocyte hypertrophy, whereas their repression reversed the Cheerio-mediated hypertrophy. Although Cheerio/Filamin B pathway mediates a mechano-protective role in the face of injury, we found excessive activity resulted in a pathological phenotype, indicating activity levels must be tightly controlled. Taken together, our data suggest that Cheerio acts via the TOR and YAP pathway to induce hypertrophic growth, as a mechano-protective response to nephrocyte injury.

A Personalized Glomerulus Chip Engineered from Stem Cell-Derived Epithelium and Vascular Endothelium

Progress in understanding kidney disease mechanisms and the development of targeted therapeutics have been limited by the lack of functional in vitro models that can closely recapitulate human physiological responses. Organ Chip (or organ-on-a-chip) microfluidic devices provide unique opportunities to overcome some of these challenges given their ability to model the structure and function of tissues and organs in vitro. Previously established organ chip models typically consist of heterogenous cell populations sourced from multiple donors, limiting their applications in patient-specific disease modeling and personalized medicine. In this study, we engineered a personalized glomerulus chip system reconstituted from human induced pluripotent stem (iPS) cell-derived vascular endothelial cells (ECs) and podocytes from a single patient. Our stem cell-derived kidney glomerulus chip successfully mimics the structure and some essential functions of the glomerular filtration barrier. We further modeled glomerular injury in our tissue chips by administering a clinically relevant dose of the chemotherapy drug Adriamycin. The drug disrupts the structural integrity of the endothelium and the podocyte tissue layers, leading to significant albuminuria as observed in patients with glomerulopathies. We anticipate that the personalized glomerulus chip model established in this report could help advance future studies of kidney disease mechanisms and the discovery of personalized therapies. Given the remarkable ability of human iPS cells to differentiate into almost any cell type, this work also provides a blueprint for the establishment of more personalized organ chip and ‘body-on-a-chip’ models in the future.

Single-Cell Profiling Reveals Transcriptional Signatures and Cell-Cell Crosstalk in Anti-PLA2R Positive Idiopathic Membranous Nephropathy Patients

Idiopathic membranous nephropathy (IMN) is an organ-specific autoimmune disease of the kidney glomerulus. It may gradually progress to end-stage renal disease (ESRD) characterized by increased proteinuria, which leads to serious consequences. Although substantial advances have been made in the understanding of the molecular bases of IMN in the last 10 years, certain questions remain largely unanswered. To define the transcriptomic landscape at single-cell resolution, we analyzed kidney samples from 6 patients with anti-PLA2R positive IMN and 2 healthy control subjects using single-cell RNA sequencing. We then identified distinct cell clusters through unsupervised clustering analysis of kidney specimens. Identification of the differentially expressed genes (DEGs) and enrichment analysis as well as the interaction between cells were also performed. Based on transcriptional expression patterns, we identified all previously described cell types in the kidney. The DEGs in most kidney parenchymal cells were primarily enriched in genes involved in the regulation of inflammation and immune response including IL-17 signaling, TNF signaling, NOD-like receptor signaling, and MAPK signaling. Moreover, cell-cell crosstalk highlighted the extensive communication of mesangial cells, which infers great importance in IMN. IMN with massive proteinuria displayed elevated expression of genes participating in inflammatory signaling pathways that may be involved in the pathogenesis of the progression of IMN. Overall, we applied single-cell RNA sequencing to IMN to uncover intercellular interactions, elucidate key pathways underlying the pathogenesis, and identify novel therapeutic targets of anti-PLA2R positive IMN.

Proteolysis and inflammation of the kidney glomerulus

Proteases play a central role in regulating renal pathophysiology and are increasingly evaluated as actionable drug targets. Here, we review the role of proteolytic systems in inflammatory kidney disease. Inflammatory kidney diseases are associated with broad dysregulations of extracellular and intracellular proteolysis. As an example of a proteolytic system, the complement system plays a significant role in glomerular inflammatory kidney disease and is currently under clinical investigation. Based on two glomerular kidney diseases, lupus nephritis, and membranous nephropathy, we portrait two proteolytic pathomechanisms and the role of the complement system. We discuss how profiling proteolytic activity in patient samples could be used to stratify patients for more targeted interventions in inflammatory kidney diseases. We also describe novel comprehensive, quantitative tools to investigate the entirety of proteolytic processes in a tissue sample. Emphasis is placed on mass spectrometric approaches that enable the comprehensive analysis of the complement system, as well as protease activities and regulation in general.

Mechanisms of Primary Membranous Nephropathy

Membranous nephropathy (MN) is an autoimmune disease of the kidney glomerulus and one of the leading causes of nephrotic syndrome. The disease exhibits heterogenous outcomes with approximately 30% of cases progressing to end-stage renal disease. The clinical management of MN has steadily advanced owing to the identification of autoantibodies to the phospholipase A2 receptor (PLA2R) in 2009 and thrombospondin domain-containing 7A (THSD7A) in 2014 on the podocyte surface. Approximately 50–80% and 3–5% of primary MN (PMN) cases are associated with either anti-PLA2R or anti-THSD7A antibodies, respectively. The presence of these autoantibodies is used for MN diagnosis; antibody levels correlate with disease severity and possess significant biomarker values in monitoring disease progression and treatment response. Importantly, both autoantibodies are causative to MN. Additionally, evidence is emerging that NELL-1 is associated with 5–10% of PMN cases that are PLA2R- and THSD7A-negative, which moves us one step closer to mapping out the full spectrum of PMN antigens. Recent developments suggest exostosin 1 (EXT1), EXT2, NELL-1, and contactin 1 (CNTN1) are associated with MN. Genetic factors and other mechanisms are in place to regulate these factors and may contribute to MN pathogenesis. This review will discuss recent developments over the past 5 years.

Collagen Deposition in Diabetic Kidney Disease Boosts Intercellular Signaling: A Mathematical Model

Diabetic kidney disease is a health burden that is becoming more prevalent in the US and worldwide. The limited options for treating and preventing diabetic kidney disease are in part due to gaps in our understanding of the progression of diabetic kidney damage and its impacts on cellular function. An important cellular function in the kidney glomerulus is intercellular communication via the release and uptake of soluble cytokines and growth factors. In diabetic kidney disease, excess collagen deposition alters the mesangial matrix properties, which, we hypothesize, diminishes the intercellular signaling between key glomerular cells. To test our hypothesis, we utilized established mathematical models of transport to study the impact of pathological deposition on the ability of cells to communicate via intercellular signaling. Our analysis reveals that pathological collagen deposition can enhance the signaling range of the glomerular cells rather than diminishing it.