Role of biophysics and mechanobiology in podocyte physiology.

Podocytes form the backbone of the glomerular filtration barrier and are exposed to various mechanical forces throughout the lifetime of an individual. The highly dynamic biomechanical environment of the glomerular capillaries greatly influences the cell biology of podocytes and their pathophysiology. Throughout the past two decades, a holistic picture of podocyte cell biology has emerged, highlighting mechanobiological signalling pathways, cytoskeletal dynamics and cellular adhesion as key determinants of biomechanical resilience in podocytes. This biomechanical resilience is essential for the physiological function of podocytes, including the formation and maintenance of the glomerular filtration barrier. Podocytes integrate diverse biomechanical stimuli from their environment and adapt their biophysical properties accordingly. However, perturbations in biomechanical cues or the underlying podocyte mechanobiology can lead to glomerular dysfunction with severe clinical consequences, including proteinuria and glomerulosclerosis. As our mechanistic understanding of podocyte mechanobiology and its role in the pathogenesis of glomerular disease increases, new targets for podocyte-specific therapeutics will emerge. Treating glomerular diseases by targeting podocyte mechanobiology might improve therapeutic precision and efficacy, with potential to reduce the burden of chronic kidney disease on individuals and health-care systems alike.

Progenitor hierarchy among parietal epithelial cells depicted at the single-cell level.

The role of parietal epithelial cells (PECs) in kidney function and disease was recently revisited. Building on previous studies of human kidney tissue, in the current issue, Liu et al. further characterize PECs using single-cell RNA sequencing data and confirm the crucial pathophysiological role of PECs in murine kidney biology as a reservoir for different types of progenitors.

USP40 deubiquitinates HINT1 and stabilizes p53 in podocyte damage.

Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.

Tissue resident cell processes determine organ damage in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects almost any organ. Multiple immunological abnormalities involving every domain of the immune system contribute to the expression of the disease. It is now recognized that elements of the immune system instigate processes in tissue resident cells which execute organ damage. Although correction of ongoing immune aberrations is important in the control of disease activity, targeting tissue specific injurious processes may prove desirable in limiting organ damage.

A comprehensive insight into autophagy and its potential signaling pathways as a therapeutic target in podocyte injury.

As part of the glomerular filtration membrane, podocyte is terminally differentiated, structurally unique, and highly specialized in maintaining kidney function. Proteinuria caused by podocyte injury (foot process effacement) is the clinical symptom of various kidney diseases (CKD), including nephrotic syndrome. Podocyte autophagy has become a powerful therapeutic strategy target in ameliorating podocyte injury. Autophagy is known to be associated significantly with sirtuin-1, proteinuria, and podocyte injury. Various key findings in podocyte autophagy were reported in the past ten years, such as the role of endoplasmic reticulum (ER) stress in podocyte autophagy impairment, podocyte autophagy-related gene, essential roles of the signaling pathways: Mammalian Target of Rapamycin (mTOR)/ Phosphoinositide 3-kinase (PI3k)/ serine/threonine kinase 1 (Akt) in podocyte autophagy. These significant factors caused podocyte injury associated with autophagy impairment. Sirtuin-1 was reported to have a vital key role in mTOR signaling, 5'AMP-activated protein kinase (AMPK) regulation, autophagy activation, and various critical pathways associated with podocyte's function and health; it has potential value to podocyte injury pathogenesis investigation. From these findings, podocyte autophagy has become an attractive therapeutic strategy to ameliorate podocyte injury, and this review will provide an in-depth review on therapeutic targets he podocyte autophagy.

Bioengineering Strategies to Develop Podocyte Culture Systems.

Unraveling the complex behavior of healthy and disease podocytes by analyzing the changes in their unique arrangement of foot processes, slit diaphragm, and the three-dimensional (3D) morphology is a long-standing goal in kidney-glomerular research. The complexities surrounding the podocytes' accessibility in animal models and growing evidence of differences between humans and animal systems have compelled researchers to look for alternate approaches to study podocyte behaviors. With the advent of bioengineered models, an increasingly powerful and diverse set of tools is available to develop novel podocyte culture systems. This review discusses the pertinence of various culture models of podocytes to study podocyte mechanisms in both normal physiology and disease conditions. While no one in vitro system comprehensively recapitulates podocytes' in vivo architecture, we emphasize how the existing systems can be exploited to answer targeted questions on podocyte structure and function. We highlight the distinct advantages and limitations of using these models to study podocyte behaviors and screen therapeutics. Finally, we discuss various considerations and potential engineering strategies for developing next-generation complex 3D culture models for studying podocyte behaviors in vitro. Impact Statement In various glomerular kidney diseases, there are numerous alterations in podocyte structure and function. Yet, many of these disease events and the required targeted therapies remain unknown, resulting in nonspecific treatments. The scientific and clinical communities actively search for new modes to develop structurally and functionally relevant podocyte culture systems to gain insights into various diseases and develop therapeutics. Current in vitro systems help in some ways but are not sufficient. A deeper understanding of these previous approaches is essential to advance the field, and importantly, bioengineering strategies can contribute a unique toolbox to establish next-generation podocyte systems.

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-ß1, 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.

Atractylodin inhibits fructose-induced human podocyte hypermotility via anti-oxidant to down-regulate TRPC6/p-CaMK4 signaling.

High fructose has been reported to drive glomerular podocyte oxidative stress and then induce podocyte foot process effacement in vivo, which could be partly regarded as podocyte hypermotility in vitro. Atractylodin possesses anti-oxidative effect. The aim of this study was to explore whether atractylodin prevented against fructose-induced podocyte hypermotility via anti-oxidative property. In fructose-exposed conditionally immortalized human podocytes, we found that atractylodin inhibited podocyte hypermotility, and up-regulated slit diaphragm proteins podocin and nephrin, and cytoskeleton protein CD2-associated protein (CD2AP), α-Actinin-4 and synaptopodin expression, which were consistent with its anti-oxidative activity evidenced by up-regulation of catalase (CAT) and superoxide dismutase (SOD) 1 expression, and reduction of reactive oxygen species (ROS) production. Atractylodin also significantly suppressed expression of transient receptor potential channels 6 (TRPC6) and phosphorylated Ca2+/calmodulin-dependent protein kinase IV (CaMK4) in cultured podocytes with fructose exposure. Additionally, in fructose-exposed podocytes, CaMK4 siRNA up-regulated synaptopodin and reduced podocyte hypermotility, whereas, silencing of TRPC6 by siRNA decreased p-CaMK4 expression, inhibited podocyte hypermotility, showing TRPC6/p-CaMK4 signaling activation in podocyte hypermotility under fructose condition. Just like atractylodin, antioxidant N-acetyl-L-cysteine (NAC) could inhibit TRPC6/p-CaMK4 signaling activation to reduce fructose-induced podocytes hypermotility. These results first demonstrated that the anti-oxidative property of atractylodin may contribute to the suppression of podocyte hypermotility via inhibiting TRPC6/p-CaMK4 signaling and restoring synaptopodin expression abnormality.

Release of HMGB1 in Podocytes Exacerbates Lipopolysaccharide-Induced Acute Kidney Injury.

OBJECTIVE: Acute kidney injury (AKI) usually occurs during sepsis. Inflammation factors, such as high-mobility group box 1 (HMGB1), are dramatically upregulated under septic conditions. In our current work, the functions of HMGB1 in AKI were explored. METHODS: An AKI model was induced by the lipopolysaccharide (LPS) challenge in C57 mice. Podocytes were challenged by LPS for different durations. Subsequently, podocytes transfected with HMGB1 siRNA were exposed to LPS for 24 h. The expressions of supernatant HMGB1 and cellular active caspase-3 were examined by Western blotting analysis. To explore the effect of HMGB1 on tubular epithelial cells (TECs), HK-2 cells were exposed to HMGB1 at various concentrations for 24 h. Epithelial-mesenchymal transition (EMT) of HK-2 cells was evaluated by Western blotting analysis. Mitochondrial division and apoptosis of HK-2 cells were assessed by MitoTracker Red and Western blotting analysis, respectively. RESULTS: Compared with the sham control group, the expression of HMGB1 was increased in the kidney of AKI mice. Moreover, the expression of supernatant HMGB1 was increased in LPS-challenged podocytes compared with the control group. Knockdown of HMGB1 attenuated LPS-induced podocyte injury. Besides, EMT in TECs was triggered by HMGB1. Mitochondrial damage and apoptosis of HK-2 cells exposed to HMGB1 were markedly elevated compared with the control group. CONCLUSIONS: Collectively, HMGB1 release in podocytes was induced by LPS, subsequently leading to exacerbated AKI.

CD73 Overexpression in Podocytes: A Novel Marker of Podocyte Injury in Human Kidney Disease.

The CD73 pathway is an important anti-inflammatory mechanism in various disease settings. Observations in mouse models suggested that CD73 might have a protective role in kidney damage; however, no direct evidence of its role in human kidney disease has been described to date. Here, we hypothesized that podocyte injury in human kidney diseases alters CD73 expression that may facilitate the diagnosis of podocytopathies. We assessed the expression of CD73 and one of its functionally important targets, the C-C chemokine receptor type 2 (CCR2), in podocytes from kidney biopsies of 39 patients with podocytopathy (including focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis (MGN) and amyloidosis) and a control group. Podocyte CD73 expression in each of the disease groups was significantly increased in comparison to controls (p < 0.001-p < 0.0001). Moreover, there was a marked negative correlation between CD73 and CCR2 expression, as confirmed by immunohistochemistry and immunofluorescence (Pearson r = -0.5068, p = 0.0031; Pearson r = -0.4705, p = 0.0313, respectively), thus suggesting a protective role of CD73 in kidney injury. Finally, we identify CD73 as a novel potential diagnostic marker of human podocytopathies, particularly of MCD that has been notorious for the lack of pathological features recognizable by light microscopy and immunohistochemistry.

REST and Stress Resistance in the Aging Kidney.

BACKGROUND: CKD is associated with the loss of functional nephr ons, leading to increased mechanical and metabolic stress in the remaining cells, particularly for cells constituting the filtration barrier, such as podocytes. The failure of podocytes to mount an adequate stress response can lead to further nephron loss and disease progression. However, the mechanisms that regulate this degenerative process in the kidney are unknown. METHODS: We combined in vitro, in vivo, and organ-on-chip approaches to identify the RE1-silencing transcription factor (REST), a repressor of neuronal genes during embryonic development, as a central regulator of podocyte adaptation to injury and aging. RESULTS: Mice with a specific deletion of REST in podocytes exhibit albuminuria, podocyte apoptosis, and glomerulosclerosis during aging, and exhibit increased vulnerability to renal injury. This phenotype is mediated, in part, by the effects of REST on the podocyte cytoskeleton that promote resistance to mechanical stressors and augment podocyte survival. Finally, REST expression is upregulated in human podocytes during aging, consistent with a conserved mechanism of stress resistance. CONCLUSIONS: These results suggest REST protects the kidney from injury and degeneration during aging, with potentially important therapeutic implications.

Single cell regulatory landscape of the mouse kidney highlights cellular differentiation programs and disease targets.

Determining the epigenetic program that generates unique cell types in the kidney is critical for understanding cell-type heterogeneity during tissue homeostasis and injury response. Here, we profile open chromatin and gene expression in developing and adult mouse kidneys at single cell resolution. We show critical reliance of gene expression on distal regulatory elements (enhancers). We reveal key cell type-specific transcription factors and major gene-regulatory circuits for kidney cells. Dynamic chromatin and expression changes during nephron progenitor differentiation demonstrates that podocyte commitment occurs early and is associated with sustained Foxl1 expression. Renal tubule cells follow a more complex differentiation, where Hfn4a is associated with proximal and Tfap2b with distal fate. Mapping single nucleotide variants associated with human kidney disease implicates critical cell types, developmental stages, genes, and regulatory mechanisms. The single cell multi-omics atlas reveals key chromatin remodeling events and gene expression dynamics associated with kidney development.

Long noncoding RNAs may impact podocytes and proximal tubule function through modulating miRNAs expression in Early Diabetic Kidney Disease of Type 2 Diabetes Mellitus patients.

Aims: Long noncoding RNAs (lncRNAs) play key roles in the pathophysiology of DKD involving actions of microRNAs (miRNAs). The aims of the study were to establish the involvement of selected lncRNAs in the epigenetic mechanisms of podocyte damage and tubular injury in DKD of type 2 diabetes mellitus (DM) patients in relation to a particular miRNAs profile. Methods: A total of 136 patients with type 2 DM and 25 healthy subjects were assessed in a cross-sectional study concerning urinary albumin: creatinine ratio (UACR), eGFR, biomarkers of podocyte damage (synaptopodin, podocalyxin) and of proximal tubule (PT) dysfunction (Kidney injury molecule-1-KIM-1, N-acetyl-D-glucosaminidase-NAG), urinary lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear-enriched abundant transcript 1 (NEAT1), myocardial infarction-associated transcript (MIAT), taurine-upregulated gene 1 (TUG1), urinary miRNA21, 124, 93, 29a. Results: Multivariable regression analysis showed that urinary lncMALAT1 correlated directly with urinary synaptopodin, podocalyxin, KIM-1, NAG, miRNA21, 124, UACR, and negatively with eGFR, miRNA93, 29a (p<0.0001; R2=0.727); urinary lncNEAT1 correlated directly with synaptopodin, KIM-1, NAG, miRNA21, 124, and negatively with eGFR, miRNA93, 29a (p<0.0001; R2=0.702); urinary lncMIAT correlated directly with miRNA93 and 29a, eGFR (p<0.0001; R2=0.671) and negatively with synaptopodin, KIM-1, NAG, UACR, miRNA21, 124 (p<0.0001; R2=0.654); urinary lncTUG1 correlated directly with eGFR, miRNA93, 29a, and negatively with synaptopodin, podocalyxin, NAG, miRNA21, 124 (p<0.0001; R2=0.748). Conclusions: In patients with type 2 DM lncRNAs exert either deleterious or protective functions within glomeruli and PT. LncRNAs may contribute to DKD through modulating miRNAs expression and activities. This observation holds true independently of albuminuria and DKD stage.

Roles of microRNAs in renal disorders related to primary podocyte dysfunction.

Through the regulation of gene expression, microRNAs (miRNAs) are capable of modulating vital biological processes, such as proliferation, differentiation, and apoptosis. Several mechanisms control the function of miRNAs, including translational inhibition and targeted miRNA degradation. Through utilizing high-throughput screening methods, such as small RNA sequencing and microarray, alterations in miRNA expression of kidneys have recently been observed both in rodent models and humans throughout the development of chronic kidney disease (CKD) and acute kidney injury (AKI). The levels of miRNAs in urine supernatant, sediment, and exosomal fraction could predict novel biomarker candidates in different diseases of kidneys, including IgA nephropathy, lupus nephritis, and diabetic nephropathy. The therapeutic potential of administrating anti-miRNAs and miRNAs has also been reported recently. The present study is aimed at reviewing the state-of-the-art research with regards to miRNAs involved in renal disorders related to primary podocyte dysfunction by laying particular emphasis on Focal Segmental Glomerulosclerosis (FSGS), Minimal Change Disease (MCD) and Membranous Nephropathy (MN).

The Basolateral Polarity Module Promotes Slit Diaphragm Formation in Drosophila Nephrocytes, a Model of Vertebrate Podocytes.

BACKGROUND: Podocyte slit diaphragms (SDs) are intercellular junctions that function as size-selective filters, excluding most proteins from urine. Abnormalities in SDs cause proteinuria and nephrotic syndrome. Podocytes exhibit apicobasal polarity, which can affect fundamental aspects of cell biology, including morphology, intercellular junction formation, and asymmetric protein distribution along the plasma membrane. Apical polarity protein mutations cause nephrotic syndrome, and data suggest apical polarity proteins regulate SD formation. However, there is no evidence that basolateral polarity proteins regulate SDs. Thus, the role of apicobasal polarity in podocytes remains unclear. METHODS: Genetic manipulations and transgenic reporters determined the effects of disrupting apicobasal polarity proteins in Drosophila nephrocytes, which have SDs similar to those of mammalian podocytes. Confocal and electron microscopy were used to characterize SD integrity after loss of basolateral polarity proteins, and genetic-interaction studies illuminated relationships among apicobasal polarity proteins. RESULTS: The study identified four novel regulators of nephrocyte SDs: Dlg, Lgl, Scrib, and Par-1. These proteins comprise the basolateral polarity module and its effector kinase. The data suggest these proteins work together, with apical polarity proteins, to regulate SDs by promoting normal endocytosis and trafficking of SD proteins. CONCLUSIONS: Given the recognized importance of apical polarity proteins and SD protein trafficking in podocytopathies, the findings connecting basolateral polarity proteins to these processes significantly advance our understanding of SD regulation.

Next-generation sequencing in patients with familial FSGS: first report of collagen gene mutations in Tunisian patients.

Focal segmental glomerulosclerosis (FSGS) is a histological lesion with many causes, including inherited genetic defects, with significant proteinuria being the predominant clinical finding at presentation. FSGS is considered as a podocyte disease due to the fact that in the majority of patients with FSGS, the lesion results from defects in the podocyte structure. However, FSGS does not result exclusively from podocyte-associated genes. In this study, we used a genetic approach based on targeted next-generation sequencing (NGS) of 242 genes to identify the genetic cause of FSGS in seven Tunisian families. The sequencing results revealed the presence of eight distinct mutations including seven newly discovered ones: the c.538G>A (p.V180M) in NPHS2, c.5186G>A (p.R1729Q) in PLCE1 and c.232A>C (p.I78L) in PAX2 and five novel mutations in COL4A3 and COL4A4 genes. Four mutations (c.209G>A (p.G70D), c.725G>A (p.G242E), c.2225G>A (p.G742E), and c. 1681_1698del) were detected in COL4A3 gene and one mutation (c.1424G>A (p.G475D)) was found in COL4A4. In summary, NGS of a targeted gene panel is an ideal approach for the genetic testing of FSGS with multiple possible underlying etiologies. We have demonstrated that not only podocyte genes but also COL4A3/4 mutations should be considered in patients with FSGS.

Apical-basal polarity regulators are essential for slit diaphragm assembly and endocytosis in Drosophila nephrocytes.

Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.

ADAM10-Mediated Ectodomain Shedding Is an Essential Driver of Podocyte Damage.

BACKGROUND: Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. METHODS: Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. RESULTS: ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. CONCLUSIONS: ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.