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Introdução: uma ótima adesão ao esquema antirretroviral de alta eficácia (HAART) é imprescindível para o sucesso terapêutico na síndrome da imunodeficiência adquirida humana (Aids). Objetivo: verificar a adesão ao HAART e identificar variáveis sociodemográficas, clínicas e laboratoriais relacionadas a esta. Métodos: estudo de inquérito simples com cálculo amostral de 81 pacientes dentre os 870 usando HAART no Serviço Especializado em HIV/Aids da Universidade Federal do Espírito Santo, Vitória – ES. Os dados foram coletados entre 14/09/2009 a 20/10/2009. Formulário padronizado e a versão traduzida e adaptada à realidade brasileira do questionário CEAT-VIH foram utilizados para acessar os dados e a adesão, respectivamente. Os desfechos foram adesão estrita (≥ 85% no CEAT-VIH) ou não, e presença ou ausência de fator possivelmente associado, utilizaram-se análises uni e multivariada (regressão logística binária) e cálculo da Odds Ratio (OR) como medida de associação. Resultados: adesão estrita foi encontrada em 42% dos pacientes, com a média de adesão de 81,1%. Falha no uso da medicação ocorreu em 76,5% dos entrevistados, cujos principais motivos foram: esquecimento (37%) e estar longe de casa (30,9%). Adesão estrita esteve estatisticamente associada a um maior número de anos completos de estudo e a um maior tempo de carga viral indetectável. Conclusão: apesar de uma média de adesão aceitável, a adesão estrita foi insatisfatória. Melhor adesão esteve associada a um maior grau de escolaridade e maior tempo de supressão virológica.

The nonmetabolizable glucose analogue [3H]-2-deoxy-D-glucose (3H-2DG) was used to study glucose transport in cultured rat podocytes. Intracellular accumulation of 3H-2DG was linear up to 20 min and was inhibited by cytochalasin B (80% inhibition) and by phlorizin (20% inhibition). Pretreatment with insulin stimulated the 3H-2DG uptake 1.5-fold. A Hill analysis of the rate of glucose transport yielded a Vmax value of approximately 10 mM and S0.5 of 7.8 mM. The value h = 1.0 for a Hill coefficient confirmed that glucose uptake exhibited a Michaelis-Menten kinetics. Transporters GLUT2 and GLUT4 were expressed in over 90% podocytes. Of the GLUT2- and GLUT4-expressing cells, approximately one-fourth expressed the membrane-bound fraction. We conclude that cultured rat podocytes possess a differentiated glucose transport system consisting chiefly of facilitative GLUT2 and GLUT4 transporters. It seems likely that a sodium-dependent glucose cotransporter may also be present in these cells.Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Deep insights into the complex cellular and molecular changes occurring during (patho-)physiological conditions are essential for understanding the interactions and regulation of proteins. This understanding is crucial for research and diagnostics. However, the effectiveness of conventional immunofluorescence and light microscope, tools for visualizing the spatial distribution of cells or proteins, are limited both in resolution and multiplexity in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, the inherent variability of samples and the optical resolution limit. The herein demonstrated combination of multiplex immunofluorescence imaging and super resolution microscopy offers a solution to these limitations by enabling the identification of different cell types and precise subcellular localization of proteins in tissue sections. In this study, we demonstrate the cyclic staining and de-staining of paraffin kidney sections, making it suitable for routine use and compatible with super-resolution microscopy for podocyte ultrastructural studies. We have further developed a computerized workflow for data processing which is accessible through available reagents and open-access code. As a proof of principle, we identified CDH2 as a marker for cellular lesions of sclerotic glomeruli in the nephrotoxic serum nephritis mouse model and cross-validated this finding with a human Nephroseq dataset indicating its translatability. In summary, our work represents an advance in multiplex imaging, which is crucial for understanding the localization of numerous proteins in a single FFPE kidney section and the compatibility with super-resolution microscopy to study ultrastructural changes of podocytes., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Identifying effective drugs for focal segmental glomerulosclerosis (FSGS) treatment holds significant importance. Our high-content drug screening on zebrafish larvae relies on nitroreductase/metronidazole (NTR/MTZ)-induced podocyte ablation to generate FSGS-like injury. A crucial factor for successful drug screenings is minimizing variability in injury induction. For this, we introduce nifurpirinol (NFP) as a more reliable prodrug for targeted podocyte depletion. NFP showed a 2.3-fold increase in efficiency at concentrations 1,600-fold lower compared with MTZ-mediated injury induction. Integration into the screening workflow validated its suitability for the high-content drug screening. The presence of crucial FSGS hallmarks, such as podocyte foot process effacement, proteinuria, and activation of parietal epithelial cells, was observed. After the isolation of the glomeruli from the larvae, we identified essential pathways by proteomic analysis. This study shows that NFP serves as a highly effective prodrug to induce the FSGS-like disease in zebrafish larvae and is well-suited for a high-content drug screening to identify new candidates for the treatment of FSGS. NEW & NOTEWORTHY This research investigated the use of nifurpirinol in nanomolar amounts as a prodrug to reliably induce focal segmental glomerulosclerosis (FSGS)-like damage in transgenic zebrafish larvae. Through proteomic analysis of isolated zebrafish glomeruli, we were further able to identify proteins that are significantly regulated after the manifestation of FSGS. These results are expected to expand our knowledge of the pathomechanism of FSGS.

Background: Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes., Methods: We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone., Results: Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria., Conclusions: Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

Mild cognitive impairment (MCI) is common in people with chronic kidney disease (CKD), and its prevalence increases with progressive loss of kidney function. MCI is characterized by a decline in cognitive performance greater than expected for an individual age and education level but with minimal impairment of instrumental activities of daily living. Deterioration can affect one or several cognitive domains (attention, memory, executive functions, language, and perceptual motor or social cognition). Given the increasing prevalence of kidney disease, more and more people with CKD will also develop MCI causing an enormous disease burden for these individuals, their relatives, and society. However, the underlying pathomechanisms are poorly understood, and current therapies mostly aim at supporting patients in their daily lives. This illustrates the urgent need to elucidate the pathogenesis and potential therapeutic targets and test novel therapies in appropriate preclinical models. Here, we will outline the necessary criteria for experimental modeling of cognitive disorders in CKD. We discuss the use of mice, rats, and zebrafish as model systems and present valuable techniques through which kidney function and cognitive impairment can be assessed in this setting. Our objective is to enable researchers to overcome hurdles and accelerate preclinical research aimed at improving the therapy of people with CKD and MCI.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease before the age of 25 yr. Nephrin, encoded by NPHS1, localizes to the slit diaphragm of glomerular podocytes and is the predominant structural component of the glomerular filtration barrier. Biallelic variants in NPHS1 can cause congenital nephrotic syndrome of the Finnish type, for which, to date, no causative therapy is available. Recently, adeno-associated virus (AAV) vectors targeting the glomerular podocyte have been assessed as a means for gene replacement therapy. Here, we established quantitative and reproducible phenotyping of a published, conditional Nphs1 knockout mouse model ( Nphs1 tm1.1Pgarg /J and Nphs2-Cre + ) in preparation for a gene replacement study using AAV vectors. Nphs1 knockout mice ( Nphs1 fl/fl Nphs2-Cre + ) exhibited 1 ) a median survival rate of 18 days (range: from 9 to 43 days; males: 16.5 days and females: 20 days); 2 ) an average foot process (FP) density of 1.0 FP/µm compared with 2.0 FP/µm in controls and a mean filtration slit density of 2.64 µm/µm 2 compared with 4.36 µm/µm 2 in controls; 3 ) a high number of proximal tubular microcysts; 4 ) the development of proteinuria within the first week of life as evidenced by urine albumin-to-creatinine ratios; and 5 ) significantly reduced levels of serum albumin and elevated blood urea nitrogen and creatinine levels. For none of these phenotypes, significant differences between sexes in Nphs1 knockout mice were observed. We quantitatively characterized five different phenotypic features of congenital nephrotic syndrome in Nphs1 fl/fl Nphs2-Cre + mice. Our results will facilitate future gene replacement therapy projects by allowing for sensitive detection of even subtle molecular effects. NEW & NOTEWORTHY To evaluate potential, even subtle molecular, therapeutic effects of gene replacement therapy (GRT) in a mouse model, prior rigorous quantifiable and reproducible disease phenotyping is necessary. Here, we, therefore, describe such a phenotyping effort in nephrin ( Nphs1 ) knockout mice to establish the basis for GRT for congenital nephrotic syndrome. We believe that our findings set an important basis for upcoming/ongoing gene therapy approaches in the field of nephrology, especially for monogenic nephrotic syndrome.

Podocyte detachment due to mechanical stress is a common issue in hypertension-induced kidney disease. This study highlights the role of zyxin for podocyte stability and function. We have found that zyxin is significantly up-regulated in podocytes after mechanical stretch and relocalizes from focal adhesions to actin filaments. In zyxin knockout podocytes, we found that the loss of zyxin reduced the expression of vinculin and VASP as well as the expression of matrix proteins, such as fibronectin. This suggests that zyxin is a central player in the translation of mechanical forces in podocytes. In vivo, zyxin is highly up-regulated in patients suffering from diabetic nephropathy and in hypertensive DOCA-salt treated mice. Furthermore, zyxin loss in mice resulted in proteinuria and effacement of podocyte foot processes that was measured by super resolution microscopy. This highlights the essential role of zyxin for podocyte maintenance in vitro and in vivo, especially under mechanical stretch., (© 2024. The Author(s).)

Podocytes, specialized postmitotic cells, are central players in various kidney-related diseases. Zebrafish have become a valuable model system for studying podocyte biology because they are genetically easy to manipulate, transparent, and their glomerular structure is similar to that of mammals. This review provides an overview of the knowledge of podocyte biology in zebrafish larvae, with particular focus on their essential contribution to understanding the mechanisms that underlie kidney diseases as well as supporting drug development. In addition, special attention is given to advances in live-imaging techniques allowing the observation of dynamic processes, including podocyte motility, podocyte process behavior, and glomerulus maturation. The review further addresses the functional aspects of podocytes in zebrafish larvae. This includes topics such as glomerular filtration, ultrastructural analyses, and evaluation of podocyte response to nephrotoxic insults. Studies presented in this context have provided important insights into the maintenance and resistance of the glomerular filtration barrier in zebrafish larvae and explored the potential transferability of these findings to mammals such as mice, rats, and most importantly, humans. The recent ability to identify potential therapeutic targets represents a promising new way to identify drugs that could effectively treat podocyte-associated glomerulopathies in humans. In summary, this review gives an overview about the importance of zebrafish as a model for podocyte-related disease and targeted drug development. It also highlights the key role of advanced imaging techniques in transparent zebrafish larvae, improving our understanding of glomerular diseases and the significant potential for translation of these findings to humans.

Background: Focal segmental glomerulosclerosis (FSGS) is a rare disease, or damage to the filtering units of the kidney, the glomeruli, about of which there is only limited knowledge and few treatment options. The STOP-FSGS consortium has set itself the goal to expand our knowledge of this disease and develop new treatment options., Project: Through intensive research and the use of state-of-the-art techniques such as super-resolution microscopy, AI-based imaging and single-cell research, the consortium aims to gain a deeper understanding of the mechanisms of FSGS. This will allow the disease to be diagnosed more accurately and thus enable targeted and more effective treatment of patients. Another focus is on the search for drugs that slow down or even cure the disease., Results: By establishing a rapid animal model, i.e. zebrafish larva, potential substances/drugs were identified that can alleviate FSGS. Moreover, super-resolution microscopy was used to precisely quantify the structural changes in the kidney by determining the so-called 'filtration slit density' (FSD) and to identify a marker allowing a personalised prognosis and assessment of the course of the disease., Conclusions: The results obtained help to better recognise the progression of FSGS and to optimally adapt treatment in order to improve the quality of life of the afflicted individuals and avoid renal replacement therapies., Competing Interests: Conflicts of interest The author declares that the technique of analysis based on super-resolution microscopy has been translated into a start-up called NIPOKA, of which she is CEO., (© Robert Koch Institute. All rights reserved unless explicitly granted.)

Chronic kidney disease is a major public health burden associated with a drastically reduced quality of living and life span that lacks suitable, individualized therapeutic strategies. Here we present a human induced pluripotent stem cell line (iPSC, UMGACBi001-A) reprogrammed from urine cells of an acute septic dialysis patient suffering from chronic kidney disease using non-integrating administration of RNAs. The generated iPSCs were positively characterized for typical morphology, pluripotency marker expression, directed differentiation potential, non-contamination, chromosomal consistency and donor identity. This iPSC-line can be a useful source for in vitro disease modelling and individualized therapeutic approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: FSGS affects the complex three-dimensional morphology of podocytes, resulting in loss of filtration barrier function and the development of sclerotic lesions. Therapies to treat FSGS are limited, and podocyte-specific drugs are unavailable. To address the need for treatments to delay or stop FSGS progression, researchers are exploring the repurposing of drugs that have been approved by the US Food and Drug Administration (FDA) for other purposes., Methods: To identify drugs with potential to treat FSGS, we used a specific zebrafish screening strain to combine a high-content screening (HCS) approach with an in vivo model. This zebrafish screening strain expresses nitroreductase and the red fluorescent protein mCherry exclusively in podocytes (providing an indicator for podocyte depletion), as well as a circulating 78 kDa vitamin D-binding enhanced green fluorescent protein fusion protein (as a readout for proteinuria). To produce FSGS-like lesions in the zebrafish, we added 80 µ M metronidazole into the fish water. We used a specific screening microscope in conjunction with advanced image analysis methods to screen a library of 138 drugs and compounds (including some FDA-approved drugs) for podocyte-protective effects. Promising candidates were validated to be suitable for translational studies., Results: After establishing this novel in vivo HCS assay, we identified seven drugs or compounds that were protective in our FSGS-like model. Validation experiments confirmed that the FDA-approved drug belinostat was protective against larval FSGS. Similar pan-histone deacetylase inhibitors also showed potential to reproduce this effect., Conclusions: Using an FSGS-like zebrafish model, we developed a novel in vivo HCS assay that identified belinostat and related pan-histone deacetylase inhibitors as potential candidates for treating FSGS., (Copyright © 2023 by the American Society of Nephrology.)

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns -/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H + symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS -/- kidney cells and injection into ctns -/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns -/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns -/- larvae, and restoration of the zebrafish pronephros function., (© 2023. The Author(s).)

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions., (© 2023. The Author(s).)

Elevation in soluble urokinase receptor (suPAR) and proteinuria are common signs in patients with moderate to severe coronavirus disease 2019 (COVID-19). Here we characterize a new type of proteinuria originating as part of a viral response. Inoculation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes increased suPAR levels and glomerulopathy in African green monkeys. Using an engineered mouse model with high suPAR expression, inhaled variants of SARS-CoV-2 spike S1 protein elicite proteinuria that could be blocked by either suPAR antibody or SARS-CoV-2 vaccination. In a cohort of 1991 COVID-19 patients, suPAR levels exhibit a stepwise association with proteinuria in non-Omicron, but not in Omicron infections, supporting our findings of biophysical and functional differences between variants of SARS-CoV-2 spike S1 protein and their binding to podocyte integrins. These insights are not limited to SARS-CoV-2 and define viral response proteinuria (VRP) as an innate immune mechanism and co-activation of podocyte integrins., (© 2023. The Author(s).)

Glomerular disease due to podocyte malfunction is a major factor in the pathogenesis of chronic kidney disease. Identification of podocyte-specific signaling pathways is therefore a prerequisite to characterizing relevant disease pathways and developing novel treatment approaches. Here, we employed loss of function studies for EPB41L5 ( Yurt ) as a central podocyte gene to generate a cell type-specific disease model. Loss of Yurt in fly nephrocytes caused protein uptake and slit diaphragm defects. Transcriptomic and proteomic analysis of human EPB41L5 knockout podocytes demonstrated impaired mechanotransduction via the YAP/TAZ signaling pathway. Further analysis of specific inhibition of the YAP/TAZ-TEAD transcription factor complex by TEADi led to the identification of ARGHAP29 as an EPB41L5 and YAP/TAZ-dependently expressed podocyte RhoGAP. Knockdown of ARHGAP29 caused increased RhoA activation, defective lamellipodia formation, and increased maturation of integrin adhesion complexes, explaining similar phenotypes caused by loss of EPB41L5 and TEADi expression in podocytes. Detection of increased levels of ARHGAP29 in early disease stages of human glomerular disease implies a novel negative feedback loop for mechanotransductive RhoA-YAP/TAZ signaling in podocyte physiology and disease.

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

SLC35F1 is a member of the sugar-like carrier (SLC) superfamily that is expressed in the mammalian brain. Malfunction of SLC35F1 in humans is associated with neurodevelopmental disorders. To get insight into the possible roles of Slc35f1 in the brain, we generated Slc35f1-deficient mice. The Slc35f1-deficient mice are viable and survive into adulthood, which allowed examining adult Slc35f1-deficient mice on the anatomical as well as behavioral level. In humans, mutation in the SLC35F1 gene can induce a Rett syndrome-like phenotype accompanied by intellectual disability (Fede et al. Am J Med Genet A 185:2238-2240, 2021). The Slc35f1-deficient mice, however, display only a very mild phenotype and no obvious deficits in learning and memory as, e.g., monitored with the novel object recognition test or the Morris water maze test. Moreover, neuroanatomical parameters of neuronal plasticity (as dendritic spines and adult hippocampal neurogenesis) are also unaltered. Thus, Slc35f1-deficient mice display no major alterations that resemble a neurodevelopmental phenotype., (© 2023. The Author(s).)

Background: For decades, knowledge about glomerular (patho)physiology has been tightly linked with advances in microscopic imaging technology. For example, the invention of electron microscopy was required to hypothesize about the mode of glomerular filtration barrier function., Summary: Super-resolution techniques, defined as fluorescence microscopy approaches that surpass the optical resolution limit of around 200 nm, have been made available to the scientific community. Several of these different techniques are currently in use in glomerular research. Using three-dimensional structured illumination microscopy, the exact morphology of the podocyte filtration slit can be morphometrically analyzed and quantitatively compared across samples originating from animal models or human biopsies., Key Messages: Several quantitative image analysis approaches and their potential influence on glomerular research and diagnostics are discussed. By improving not only optical resolution but also information content and turnaround time, super-resolution microscopy has the potential to expand the diagnosis of glomerular disease. Soon, these approaches could be introduced into glomerular disease diagnosis., Competing Interests: Florian Siegerist holds shares, Vedran Drenic and Nihal Telli are employees, and Nicole Endlich serves as CEO and holds shares of NIPOKA GmbH, Greifswald, Germany, a startup commercializing PEMP. Thor-Magnus Koppe has no conflicts of interest., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Given the increasing prevalence of chronic kidney disease (CKD) and its impact on health care, it is important to better understand the multiple factors influencing health-related quality of life (HRQOL), particularly since they have been shown to affect CKD outcomes. Determinants of HRQOL as measured by the validated Kidney Disease Quality of Life questionnaire (KDQOL) and the Patient Health Questionnaire depression screener (PHQ-9) were assessed in a routine CKD patient sample, the Greifswald Approach to Individualized Medicine (GANI_MED) renal cohort (N = 160), including a wide range of self-reported data, sociodemographic and laboratory measures. Compared to the general population, CKD patients had lower HRQOL indices. Dialysis was associated with (1) low levels of physical functioning, (2) increased impairments by symptoms and problems, and (3) more effects and burden of kidney disease. HRQOL is seriously affected in CKD patients. However, impairments were found irrespective of eGFR decline and albuminuria. Rather, the comorbid conditions of depression and diabetes predicted a lower HRQOL (physical component score). Further studies should address whether recognizing and treating depression may not only improve HRQOL but also promote survival and lower hospitalization rates of CKD patients.

Background : Obesity, hyperglycemia and hypertension are critical risk factors for development of diabetic kidney disease (DKD). Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular and renal outcomes in type 2 diabetes patients. Here, we characterized the effect of the long-acting GLP-1R agonist semaglutide alone and in combination with an ACE inhibitor (lisinopril) in a model of hypertension-accelerated, advanced DKD facilitated by adeno-associated virus-mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) female diabetic db / db mice. Methods : Female db / db mice received a single intravenous injection of ReninAAV 1 week prior to UNx. Six weeks post-nephrectomy, db / db UNx-ReninAAV mice were administered (q.d.) vehicle, semaglutide (30 nmol/kg, s.c.) or semaglutide (30 nmol/kg, s.c.) + lisinopril (30 mg/kg, p.o.) for 11 weeks. Endpoints included blood pressure, plasma/urine biochemistry, kidney histopathology and RNA sequencing. Results : Vehicle-dosed db / db UNx-ReninAAV mice developed hallmarks of DKD characterized by severe albuminuria and advanced glomerulosclerosis. Semaglutide robustly reduced hyperglycemia, hypertension and albuminuria concurrent with notable improvements in glomerulosclerosis severity, podocyte filtration slit density, urine/renal kidney injury molecule-1 (KIM-1) levels and gene expression markers of inflammation and fibrogenesis in db / db UNx-ReninAAV mice. Co-administration of lisinopril further ameliorated hypertension and glomerulosclerosis. Conclusions : Semaglutide improves disease hallmarks in the db / db UNx-ReninAAV mouse model of advanced DKD. Further benefits on renal outcomes were obtained by adjunctive antihypertensive standard of care. Collectively, our study supports the development of semaglutide for management of DKD.

Background: Most patients with chronic kidney disease (CKD) are old, comorbid, and subjected to polypharmacy. This study describes prevalence and predictors of potentially inappropriate medication (PIM) in CKD patients., Materials and Methods: Medication plans of CKD patients of the "Greifswald Approach to Individualized Medicine" cross-sectional study (GANI_MED) were checked for PIM based on kidney function (PIM-K) and PIM for elderly patients (PIM-E). PIM-K were defined by prescription instructions of product labeling. PIM-E were defined by BEERS, -PRISCUS, and FORTA criteria. Predictors for PIM were identified through multiple stepwise regression., Results: 375 patients were included (age: 67.9 ± 13.5 years; estimated glomerular filtration rate (eGFR): 23.3 ± 18.6 mL/min/1.73m 2 ; prescriptions: 11.1 ± 4.7). 44.5% of all CKD patients had PIM-K, and 43.2 to 79.0% of all elderly patients had PIM-E. Polypharmacy and reduced eGFR were predictors for PIM. The risk for PIM-K was increased by 3.8 (95% confidence interval (CI): 1.5 - 9.6) with 10 or more prescriptions and by 8.7 (95% CI: 1.3 - 58.5) with an eGFR below 30 mL/min/1.73m 2 . On average, elderly patients with 10 or more prescriptions had 3.0 ± 1.7 PIM-E., Conclusion: Polypharmacy, PIM-K, and PIM-E affect many CKD patients and can lead to adverse events. Deprescribing and targeted prescribing may improve the outcome of CKD patients and elderly patients.

Calcimimetic agents allosterically increase the calcium ion sensitivity of the calcium-sensing receptor (CaSR), which is expressed in the tubular system and to a lesser extent in podocytes. Activation of this receptor can reduce glomerular proteinuria and structural damage in proteinuric animal models. However, the precise role of the podocyte CaSR remains unclear. Here, a CaSR knockdown in cultured murine podocytes and a podocyte-specific CaSR knockout in BALB/c mice were generated to study its role in proteinuria and kidney function. Podocyte CaSR knockdown abolished the calcimimetic R-568 mediated calcium ion-influx, disrupted the actin cytoskeleton, and reduced cellular attachment and migration velocity. Adriamycin-induced proteinuria enhanced glomerular CaSR expression in wild-type mice. Albuminuria, podocyte foot process effacement, podocyte loss and glomerular sclerosis were significantly more pronounced in adriamycin-treated podocyte-specific CaSR knockout mice compared to wild-type littermates. Co-treatment of wild-type mice with adriamycin and the calcimimetic cinacalcet reduced proteinuria in wild-type, but not in podocyte-specific CaSR knockout mice. Additionally, four children with nephrotic syndrome, whose parents objected to glucocorticoid therapy, were treated with cinacalcet for one to 33 days. Proteinuria declined transiently by up to 96%, serum albumin increased, and edema resolved. Thus, activation of podocyte CaSR regulates key podocyte functions in vitro and reduced toxin-induced proteinuria and glomerular damage in mice. Hence, our findings suggest a potential novel role of CaSR signaling in control of glomerular disease., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single-cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to obtain spatial single-cell single-mRNA expression data from routinely generated kidney biopsies. Deep learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single-cell expression data were assigned. Furthermore, we show correlative and spatial single-cell expression quantification with super-resolved podocyte foot process morphometry. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single-cell level with single-mRNA resolution. Using this method, we demonstrate that ACE2 can be locally upregulated in podocytes upon injury. In a patient suffering from COVID-19-associated collapsing FSGS, ACE2 expression levels were correlated with intracellular SARS-CoV-2 abundance. As this method performs well with standard formalin-fixed paraffin-embedded samples and we provide pretrained deep learning networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse Glom Assay as a semi-automated high-throughput screening method-shGlom Assay -allowing the analysis of several hundreds of FDA-verified compounds in combination with downstream pathway analysis like transcriptomic and proteomic analyses from the same samples, using a small number of animals. In an initial prescreening we have identified vitamin D3 and its analog calcipotriol to be protective on podocytes. Furthermore, by using RT-qPCR, Western blot, and RNA sequencing, we found that mRNA and protein expression of nephrin, the vitamin D receptor and specific podocyte markers were significantly up-regulated due to vitamin D3- and calcipotriol-treatment. In contrast, kidney injury markers were significantly down-regulated. Additionally, we found that vitamin D3 and calcipotriol have had neither influence on the expression of the miR-21 and miR-30a nor on miR-125a/b, a miRNA described to regulate the vitamin D receptor. In summary, we advanced the established mouse Glom Assay to a semi-automated high-throughput assay and combined it with downstream analysis techniques by using only a minimum number of animals. Hereby, we identified the vitamin D signaling pathway as podocyte protective and to be counteracting their de-differentiation., Competing Interests: JG is an employee of the DITABIS AG, Pforzheim, Germany and the ACQUIFER Imaging GmbH, Heidelberg, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ristov, Lange, Artelt, Nath, Kuss, Gehrig, Lindenmeyer, Cohen, Gul, Endlich, Völker and Endlich.)

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.

Background: The cell-matrix adhesion between podocytes and the glomerular basement membrane is essential for the integrity of the kidney's filtration barrier. Despite increasing knowledge about the complexity of integrin adhesion complexes, an understanding of the regulation of these protein complexes in glomerular disease remains elusive., Methods: We mapped the in vivo composition of the podocyte integrin adhesome. In addition, we analyzed conditional knockout mice targeting a gene ( Parva ) that encodes an actin-binding protein (α-parvin), and murine disease models. To evaluate podocytes in vivo , we used super-resolution microscopy, electron microscopy, multiplex immunofluorescence microscopy, and RNA sequencing. We performed functional analysis of CRISPR/Cas9-generated PARVA single knockout podocytes and PARVA and P ARVB double knockout podocytes in three- and two-dimensional cultures using specific extracellular matrix ligands and micropatterns., Results: We found that PARVA is essential to prevent podocyte foot process effacement, detachment from the glomerular basement membrane, and the development of FSGS. Through the use of in vitro and in vivo models, we identified an inherent PARVB -dependent compensatory module at podocyte integrin adhesion complexes, sustaining efficient mechanical linkage at the filtration barrier. Sequential genetic deletion of PARVA and PARVB induces a switch in structure and composition of integrin adhesion complexes. This redistribution of these complexes translates into a loss of the ventral actin cytoskeleton, decreased adhesion capacity, impaired mechanical resistance, and dysfunctional extracellular matrix assembly., Conclusions: The findings reveal adaptive mechanisms of podocyte integrin adhesion complexes, providing a conceptual framework for therapeutic strategies to prevent podocyte detachment in glomerular disease., (Copyright © 2022 by the American Society of Nephrology.)

Vaccine-induced immune thrombotic thrombocytopenia (VITT; synonym, thrombosis with thrombocytopenia syndrome, is associated with high-titer immunoglobulin G antibodies directed against platelet factor 4 (PF4). These antibodies activate platelets via platelet FcγIIa receptors, with platelet activation greatly enhanced by PF4. Here we summarize the current concepts in the pathogenesis of VITT. We first address parallels between heparin-induced thrombocytopenia and VITT, and provide recent findings on binding of PF4 to adenovirus particles and non-assembled adenovirus proteins in the 2 adenovirus vector-based COVID-19 vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S. Further, we discuss the potential role of vaccine constituents such as glycosaminoglycans, EDTA, polysorbate 80, human cell-line proteins and nucleotides as potential binding partners of PF4. The immune response towards PF4 in VITT is likely triggered by a proinflammatory milieu. Human cell-line proteins, non-assembled virus proteins, and potentially EDTA may contribute to the proinflammatory state. The transient nature of the immune response towards PF4 in VITT makes it likely that-as in heparin-induced thrombocytopenia -marginal zone B cells are key for antibody production. Once high-titer anti-PF4 antibodies have been formed 5 to 20 days after vaccination, they activate platelets and granulocytes. Activated granulocytes undergo NETosis and the released DNA also forms complexes with PF4, which fuels the Fcγ receptor-dependent cell activation process, ultimately leading to massive thrombin generation. Finally, we summarize our initial observations indicating that VITT-like antibodies might also be present in rare patients with recurrent venous and arterial thrombotic complications, independent of vaccination., Competing Interests: Conflicts of interest Dr Schönborn is the recipient of a young investigator grant of the medical faculty of the Universitätsmedizin Greifswald. Dr. Greinacher reports grants and non-financial support from Aspen, Boehringer Ingelheim, MSD, Bristol Myers Squibb (BMS), Paringenix, Bayer Healthcare, Gore Inc., Rovi, Sagent, Biomarin/Prosensa, personal fees from Aspen, Boehringer Ingelheim, MSD, Macopharma, BMS, Chromatec, Instrumentation Laboratory, nonfinancial support from Boehringer Ingelheim, Portola, Ergomed, GTH e.V. outside the submitted work. Dr. Thiele reports personal fees from Bristol Myers Squibb, Bayer, Daichii Sankyo, Pfizer, Novo Nordisk, Chugai Pharma, and Novartis, all of which are outside of the submitted manuscript. None of the other authors has to declare a conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

In the glomerulus, Bowman's space is formed by a continuum of glomerular epithelial cells. In focal segmental glomerulosclerosis (FSGS), glomeruli show segmental scarring, a result of activated parietal epithelial cells (PECs) invading the glomerular tuft. The segmental scars interrupt the epithelial continuum. However, non-sclerotic segments seem to be preserved even in glomeruli with advanced lesions. We studied the histology of the segmental pattern in Munich Wistar Frömter rats, a model for secondary FSGS. Our results showed that matrix layers lined with PECs cover the sclerotic lesions. These PECs formed contacts with podocytes of the uninvolved tuft segments, restoring the epithelial continuum. Formed Bowman's spaces were still connected to the tubular system. In biopsies of patients with secondary FSGS, we also detected matrix layers formed by PECs, separating the uninvolved from the sclerotic glomerular segments. PECs have a major role in the formation of glomerulosclerosis; we show here that in FSGS they also restore the glomerular epithelial cell continuum that surrounds Bowman's space. This process may be beneficial and indispensable for glomerular filtration in the uninvolved segments of sclerotic glomeruli., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Kidney function has two important elements: glomerular filtration and tubular function (secretion and reabsorption). A persistent decrease in glomerular filtration rate (GFR), with or without proteinuria, is diagnostic of chronic kidney disease (CKD). While glomerular injury or disease is a major cause of CKD and usually associated with proteinuria, predominant tubular injury, with or without tubulointerstitial disease, is typically non-proteinuric. CKD has been linked with cognitive impairment, but it is unclear how much this depends on a decreased GFR, altered tubular function or the presence of proteinuria. Since CKD is often accompanied by tubular and interstitial dysfunction, we explore here for the first time the potential role of the tubular and tubulointerstitial compartments in cognitive dysfunction. To help address this issue we selected a group of primary tubular diseases with preserved GFR in which to review the evidence for any association with brain dysfunction. Cognition, mood, neurosensory and motor disturbances are not well characterized in tubular diseases, possibly because they are subclinical and less prominent than other clinical manifestations. The available literature suggests that brain dysfunction in tubular and tubulointerstitial diseases is usually mild and is more often seen in disorders of water handling. Brain dysfunction may occur when severe electrolyte and water disorders in young children persist over a long period of time before the diagnosis is made. We have chosen Bartter and Gitelman syndromes and nephrogenic diabetes insipidus as examples to highlight this topic. We discuss current published findings, some unanswered questions and propose topics for future research., (© The Author(s) 2021. Published by Oxford University Press on behalf of the ERA.)

Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background., Competing Interests: The authors have declared that no competing interests exist.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia., (© 2021 by The American Society of Hematology.)

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies., (© 2021. The Author(s).)

High salt diet (HSD) is a hallmark of blood pressure elevations, weight gain and diabetes onset in the metabolic syndrome. In kidney, compensatory mechanisms are activated to balance salt turnover and maintain homeostasis. Data on the long-term effects of HSD with respect to tubular cell functions and kidney architecture that exclude confounding indirect blood pressure effects are scarce. Additionally we focus on cold shock Y-box binding protein-1 as a tubular cell protective factor. A HSD model (4% NaCl in chow; 1% NaCl in water) was compared to normal salt diet (NSD, standard chow) over 16 months using wild type mice and an inducible conditional whole body knockout for cold shock Y-box binding protein-1 (BL6J/N, Ybx1). HSD induced no difference in blood pressure over 16 months, comparing NSD/HSD and Ybx1 wild type/knockout. Nevertheless, marked phenotypic changes were detected. Glucosuria and subnephrotic albuminuria ensued in wild type animals under HSD, which subsided in Ybx1-deficient animals. At the same time megalin receptors were upregulated. The sodium-glucose cotransporter-2 (SGLT2) was completely downregulated in wild type HSD animals that developed glucosuria. In Ybx1 knockouts, expression of AQP1 and SGLT2 was maintained under HSD; proximal tubular widening and glomerular tubularization developed. Concurrently, amino aciduria of neutral and hydrophobic amino acids was seen. In vitro translation confirmed that YB-1 translationally represses Sglt2 transcripts. Our data reveal profound effects of HSD primarily within glomeruli and proximal tubular segments. YB-1 is regulated by HSD and orchestrates HSD-dependent changes; notably, sets reabsorption thresholds for amino acids, proteins and glucose., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D-structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin-5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background/aims: Podocyte differentiation is essential for proper blood filtration in the kidney. It is well known that transcription factors play an essential role to maintain the differentiation of podocytes. The present study is focused on the basic helix-loop-helix (bHLH) transcription factor Tcf21 (Pod1) which is essential for the development of podocytes in vivo. Since parietal epithelial cells (PECs) are still under debate to be progenitor cells which can differentiate into podocytes, we wanted to find out whether the expression of Tcf21 induces a transition of PECs into podocytes., Methods: We transfected PECs with Tcf21-GFP and analyzed the expression of PEC- and podocyte-specific markers. Furthermore, we performed ChIP-Seq analysis to identify new putative interaction partners and target genes of Tcf21., Results: By gene arrays analysis, we found that podocytes express high levels of Tcf21 in vivo in contrast to cultured podocytes and parietal epithelial cells (PECs) in vitro. After the expression of Tcf21 in PECs, we observed a downregulation of specific PEC markers like caveolin‑1, β-catenin and Pax2. Additionally, we found that the upregulation of Tcf21 induced multi-lobulation of cell nuclei, budding and a formation of micronuclei (MBM). Furthermore, a high number of PECs showed a tetraploid set of chromosomes. By qRT-PCR and Western blot analysis, we revealed that the transcription factor YY1 is downregulated by Tcf21. Interestingly, co-expression of YY1 and Tcf21 rescues MBM and reduced tetraploidy. By ChIP-Seq analysis, we identified a genome-wide Tcf21-binding site (CAGCTG), which matched the CANNTG sequence, a common E-box binding motif used by bHLH transcription factors. Using this technique, we identified additional Tcf21 targets genes that are involved in the regulation of the cell cycle (e.g. Mdm2, Cdc45, Cyclin D1, Cyclin D2), on the stability of microtubules (e.g. Mapt) as well as chromosome segregation., Conclusion: Taken together, we demonstrate that Tcf21 inhibits the expression of PEC-specific markers and of the transcription factor YY1, induces MBM as well as regulates the cell cycle suggesting that Tcf21 might be important for PEC differentiation into podocyte-like cells., Competing Interests: The authors declare no competing financial interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Glomerular hypertension induces mechanical load to podocytes, often resulting in podocyte detachment and the development of glomerulosclerosis. Although it is well known that podocytes are mechanosensitive, the mechanosensors and mechanotransducers are still unknown. Since filamin A, an actin-binding protein, is already described to be a mechanosensor and mechanotransducer, we hypothesized that filamins could be important for the outside-in signaling as well as the actin cytoskeleton of podocytes under mechanical stress. In this study, we demonstrate that filamin A is the main isoform of the filamin family that is expressed in cultured podocytes. Together with filamin B, filamin A was significantly up-regulated during mechanical stretch (3 days, 0.5 Hz, and 5% extension). To study the role of filamin A in cultured podocytes under mechanical stress, filamin A was knocked down (Flna KD) by specific siRNA. Additionally, we established a filamin A knockout podocyte cell line (Flna KO) by CRISPR/Cas9. Knockdown and knockout of filamin A influenced the expression of synaptopodin, a podocyte-specific protein, focal adhesions as well as the morphology of the actin cytoskeleton. Moreover, the cell motility of Flna KO podocytes was significantly increased. Since the knockout of filamin A has had no effect on cell adhesion of podocytes during mechanical stress, we simultaneously knocked down the expression of filamin A and B. Thereby, we observed a significant loss of podocytes during mechanical stress indicating a compensatory mechanism. Analyzing hypertensive mice kidneys as well as biopsies of patients suffering from diabetic nephropathy, we found an up-regulation of filamin A in podocytes in contrast to the control. In summary, filamin A and B mediate matrix-actin cytoskeleton interactions which are essential for the adaptation of cultured podocyte to mechanical stress., (© 2021 Federation of American Societies for Experimental Biology.)