Nephroprotective and antioxidant effects of ethanol extract of Coprinus comatus mushroom fruit-bodies on streptozotocin-induced diabetic rat models

Diabetes mellitus (DM) is a metabolic disease with an increasing prevalence. An increase in free radicals and AGEs can cause glomerular cell damage which can lead to inflammation and DM nephropathy. WHO has recommended DM treatment by using herbal medicines that have minimal side effects and have a lot of biological activity to prevent complications. Coprinus comatus (O.F. Mull.) is known as immunomodulatory, anti-inflammatory and antidiabetic agent. The research used C. comatus cultivated in Cianjur. The basidiome of this mushroom has slightly oval and small with height of 8-12 cm and thickness of 2-3 cm. The research included six groups of male Wistar rats: Group 1 received no treatment, Groups 2–6 were administered 45 mg/kg BW streptozotocin once, Group 3 was administered 45 mg/kg BW metformin, Groups 4, 5, and 6 were administered 250, 500, and 750 mg/kg BW of C. comatus fruit body ethanol extract respectively for 14 days. The superoxide dismutase (SOD), malondialdehyde (MDA), hepcidin and β2 microglobulin (B2M) levels were evaluated. Data were analyzed using analysis of variance and Duncan’s multiple-range tests. The results showed that dose of 500 mg was effective in increasing SOD and decreasing hepcidin, B2M and MDA levels.

EFFECTS OF CORYNEBACTERIUM PSEUDOTUBERCULOSIS ON SHEEP KIDNEYS

Light and electron microscopic examinations were performed on kidneys of sheep infected with caseous lymphadenitis ( CLA ) caused by Corynebacterium pseudotuberculosis Membranproliferative glomerulonephritis was demonstrated. There were irregular thickenings of the glomeruelar basement membrane and glomerular cell proliferation, in contrast, no such lesions were found in the control lambs, indicating that CLA play a role in inducing glomerulorephritis

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor β (TGFβ), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1β (IL-1β) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

New Endothelial Mechanisms in Glomerular (Patho)biology and Proteinuria Development Captured by Intravital Multiphoton Imaging

In the past two decades, intravital imaging using multiphoton microscopy has provided numerous new visual and mechanistic insights into glomerular biology and disease processes including the function of glomerular endothelial cells (GEnC), podocytes, and the development of proteinuria. Although glomerular endothelial injury is known to precede podocyte damage in several renal diseases, the primary role of GEnCs in proteinuria development received much less attention compared to the vast field of podocyte pathobiology. Consequently, our knowledge of GEnC mechanisms in glomerular diseases is still emerging. This review highlights new visual clues on molecular and cellular mechanisms of GEnCs and their crosstalk with podocytes and immune cells that were acquired recently by the application of multiphoton imaging of the intact glomerular microenvironment in various proteinuric disease models. New mechanisms of glomerular tissue remodeling and regeneration are discussed based on results of tracking the fate and function of individual GEnCs using serial intravital multiphoton imaging over several days and weeks. The three main topics of this review include (i) the role of endothelial injury and microthrombi in podocyte detachment and albumin leakage via hemodynamic and mechanical forces, (ii) the alterations of the endothelial surface layer (glycocalyx) and its interactions with circulating immune cells in lupus nephritis, and (iii) the structural and functional remodeling and regeneration of GEnCs in hypertension, diabetes, and other experimental injury conditions. By the comprehensive visual portrayal of GEnCs and the many other contributing glomerular cell types, this review emphasizes the complexity of pathogenic mechanisms that result in proteinuria development.

The Histomorphometry of Liver and Kidney of Hyperglycemic Albino Rats after Treatment with Tithonia diversifolia Leaf Extract

Tithonia diversifolia can be used as an antidiabetic, so it is necessary to study the safety of its use, especially the side effects on the liver and kidneys. This study aimed to determine the effect of using T. diversifolia leaf extract through histomorphometry observations of the liver and kidneys of hyperglycemic albino rats. The study design used a completely randomized design (CRD). This study used 20 male albino rats which were divided into five treatment groups, there were T0 (rat normal/control), T1 (hyperglycemic rat without T. diversifolia leaf extract), T2 (hyperglycemic rat administered with Glibenclamide 10 mg/kg BW), T3 (hyperglycemic rat administered with T. diversifolia leaf extract 150 mg/kg BW), T4 (hyperglycemic rat administered with T. diversifolia leaf extract 300 mg/kg BW). Every treatment was repeated four times. The damage of hepatocyte and the glomerular cell was observed through histological structure observation by histomorphometry method using a photomicrography microscope (Olympus BX51). The results indicate that there were significant differences (P < 0.05) in the variable of hepatocytes diameter and there was no significant different (P > 0.05) result on glomerular diameter, as well as kidney and liver weight. It was concluded that T. diversifolia leaf extract of 150 mg/kg BW and 300 mg/kg BW are safe to be used as antidiabetic. It does not cause any side effects on the liver and kidneys of hyperglycemic albino rats. Thereby T. diversifolia leaf extract can be further tested as preparation of biopharmaca which can be used as herbal medicines for diabetics.

Single Cell Transcriptome Helps Better Understanding Crosstalk in Diabetic Kidney Disease

Years of research revealed that crosstalk extensively existed among kidney cells, cell factors and metabolites and played an important role in the development of diabetic kidney disease (DKD). In the last few years, single-cell RNA sequencing (scRNA-seq) technology provided new insight into cellular heterogeneity and genetic susceptibility regarding DKD at cell-specific level. The studies based on scRNA-seq enable a much deeper understanding of cell-specific processes such as interaction between cells. In this paper, we aim to review recent progress in single cell transcriptomic analyses of DKD, particularly highlighting on intra- or extra-glomerular cell crosstalk, cellular targets and potential therapeutic strategies for DKD.

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice

Current interventions are not effectives in preventing sepsis-induced acute kidney injury and its long-term outcomes or even after second renal insult. Therapeutic strategies using lipid mediators, as aspirin-triggered resolvin D1 (ATRvD1), can contribute for resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing the proteinuria excretion, UPC ratio, glomerular cell number and extracellular matrix deposition. Pro-fibrotic markers, as transforming growth factor &beta; (TGFb), type 3 collagen and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from renal apoptosis and recruitment of F4/80+ cells. Interleukin-1b (IL-1b) levels were increased in subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-a (TNF-a), IL-10 and IL-4 mRNA expression was increased in the kidney of BSA-challenged post-septic mice and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

Tripartite Separation of Glomerular Cell-Types and Proteomes From Reporter-Free Mice

Background: The glomerulus comprises podocytes, mesangial, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell-types for biochemical investigations. We developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). Methods: Glomerular cell-types were separated via a novel FACS-sort approach from wildtype or mT/mG mice and the purity validated. Cell-type proteomes were compared between strains, ages, and sex. TimMEP was applied to the podocyte-targeting immunologic THSD7A-associated membranous nephropathy model. Results: TimMEP enabled protein-biochemical analyses of podocytes, mesangial, and endothelial cells derived from reporter-free mice and allowed the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. Marker proteins for mesangial and endothelial proteins were identified and protein-based potential communication networks and phosphorylation patterns outlined. The analysis detected cell-type specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response chiefly in podocytes, which bulk glomerular analyses could not detect. Combination of proteomics with super-resolution imaging revealed a specific loss of slit-diaphragm but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model. Conclusion: TimMEP enables glomerular cell-type resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way towards the comprehensive and systematic characterization of glomerular cell-type biology.

MO005GENERATION OF NOVEL 3D CO-CULTURE SYSTEMS TO STUDY PODOCYTOPATHIES EX VIVO IN A PERSONALIZED MANNER*

Background and Aims The smallest filtration unit of the kidney, the glomerulus, is composed of capillaries formed by glomerular endothelial cells (GEC), glomerular basement membrane and podocytes. Furthermore, glomerular mesangial cells (GMC) between the capillary loops give structural support. It is known that loss of podocyte foot processes is leading to a dysfunctional glomerular filtration barrier and is seen in glomerular diseases like focal segmental glomerulosclerosis and other podocytopathies. Indeed, it is hard to investigate podocytes in culture because podocytes are terminally end-differentiated cells that do not proliferate, lack foot processes and cell type-specific markers. Although conditionally immortalized human podocytes regained the capacity of proliferation, marker expression and behaviour differ between cell lines. The overarching aims of this study are to generate a 3D glomerular co-culture model that better reflects the in vivo phenotype of glomerular cell types. We want to investigate cell-cell contact, interaction and communication and extracellular matrix production in 3D glomerular co-cultures. Furthermore, patient-derived hiPSC-podocytes will be used in the glomerular co-cultures to investigate podocyte disease in a personalized manner and to identify potential therapeutic targets. Method The hanging droplet method was used to produce 3D glomerular spheroids. Therefore, human differentiated immortalized podocytes, human GECs and human GMCs were inserted in a medium-droplet hanging from the lid of a petri dish and harvested at different time points. Fluorescent cell lines of the different glomerular cell types were tracked in a time-lapse experiment to study if cell attachment and spheroid formation undergoes a specific order and structure. Glomerular spheroids were further characterized regarding the expression of podocyte-specific markers and extracellular matrix synthesis by immunohistochemistry, electron microscopy and qPCR and were compared to human cells isolated from glomeruli. Furthermore, scRNA-sequencing analysis was performed in 2D mono-cultures of human GECs, GMCs and immortalized podocytes and on 3D co-cultures to see if this change in culture conditions leads to transcriptomic alterations. For the generation of patient-derived podocytes, skin fibroblast of patients with podocyte mutations (INF2 mutation and WT1 mutation) and from healthy controls were reprogramed in iPSCs and differentiated into podocytes that keep the patient’s mutation. Results First time-lapse experiments of glomerular co-cultures showed that human podocytes and human glomerular endothelial cells attach to each other (Fig. 1a) and histological sections revealed that the glomerular spheroids are encapsulated by a monolayer of cells (Fig. 1b). SEM allowed ultrastructural characterization of the 3D spheroid-like structures (Fig. 1c). TEM revealed cell protrusions of podocytes that were not seen in monocultures (Fig. 1d, e). We could also demonstrate production of extracellular matrix by the cells (Fig. 1f). Immunohistochemistry and qPCR showed expression of collagen-IV and laminin. During the reprogramming of patient-derived fibroblasts, size of the generated hiPSC decreased and the nuclei to cell body ratio increased. HiPSCs formed colonies with distinct boarders and the proliferation rate increased. Furthermore, generated hiPSC showed similar gene expression of pluripotency markers compared to a commercial hiPSC control cell line and podocytes derived from these hiPSC expressed synaptopodin (Fig. 2). Conclusion We generated a 3D co-culture model that better represents the complexity of the glomerulus ex vivo. It is indicated that this model provides better physiological conditions. By an insertion of patient-specific hiPSC-derived podocytes in the 3D co-culture we will investigate glomerular diseases in a personalized manner in the future.

Single-cell RNA sequencing reveals the mesangial identity and species diversity of glomerular cell transcriptomes

Molecular characterization of the individual cell types in human kidney as well as model organisms are critical in defining organ function and understanding translational aspects of biomedical research. Previous studies have uncovered gene expression profiles of several kidney glomerular cell types, however, important cells, including mesangial (MCs) and glomerular parietal epithelial cells (PECs), are missing or incompletely described, and a systematic comparison between mouse and human kidney is lacking. To this end, we use Smart-seq2 to profile 4332 individual glomerulus-associated cells isolated from human living donor renal biopsies and mouse kidney. The analysis reveals genetic programs for all four glomerular cell types (podocytes, glomerular endothelial cells, MCs and PECs) as well as rare glomerulus-associated macula densa cells. Importantly, we detect heterogeneity in glomerulus-associated Pdgfrb-expressing cells, including bona fide intraglomerular MCs with the functionally active phagocytic molecular machinery, as well as a unique mural cell type located in the central stalk region of the glomerulus tuft. Furthermore, we observe remarkable species differences in the individual gene expression profiles of defined glomerular cell types that highlight translational challenges in the field and provide a guide to design translational studies.