Tubuloid culture enables long-term expansion of functional human kidney tubule epithelium from iPSC-derived organoids.

Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting 'iPSC organoid-derived (iPSCod)' tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling.

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.

PDGF-D Is Dispensable for the Development and Progression of Murine Alport Syndrome.

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. In vitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3-/- mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3-/- mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3-/- mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3-/-Pdgfd-/- mice compared with Col4a3-/-Pdgfd+/+ littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3-/- mice might explain the lack of effects in vivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.

Human scattered tubular cells represent a heterogeneous population of glycolytic dedifferentiated proximal tubule cells.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13+ CD24- CD133- proximal tubule epithelial cells (PTECs) and CD13+ CD24+ and CD13+ CD133+ STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor κB, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The Role of Platelet-Derived Growth Factor in Focal Segmental Glomerulosclerosis.

BACKGROUND: FSGS is the final common pathway to nephron loss in most forms of severe or progressive glomerular injury. Although podocyte injury initiates FSGS, parietal epithelial cells (PECs) are the main effectors. Because PDGF takes part in fibrotic processes, we hypothesized that the ligand PDGF-B and its receptor PDGFR- ß participate in the origin and progression of FSGS. METHODS: We challenged Thy1.1 transgenic mice, which express Thy1.1 in the podocytes, with anti-Thy1.1 antibody to study the progression of FSGS. We investigated the role of PDGF in FSGS using challenged Thy1.1 mice, 5/6 nephrectomized mice, Col4 -/- (Alport) mice, patient kidney biopsies, and primary murine PECs, and challenged Thy1.1 mice treated with neutralizing anti-PDGF-B antibody therapy. RESULTS: The unchallenged Thy1.1 mice developed only mild spontaneous FSGS, whereas challenged mice developed progressive FSGS accompanied by a decline in kidney function. PEC activation, proliferation, and profibrotic phenotypic switch drove the FSGS. During disease, PDGF-B was upregulated in podocytes, whereas PDGFR- ß was upregulated in PECs from both mice and patients with FSGS. Short- and long-term treatment with PDGF-B neutralizing antibody improved kidney function and reduced FSGS, PEC proliferation, and profibrotic activation. In vitro , stimulation of primary murine PECs with PDGF-B recapitulated in vivo findings with PEC activation and proliferation, which was inhibited by PDGF-B antibody or imatinib. CONCLUSION: PDGF-B-PDGFR- ß molecular crosstalk between podocytes and PECs drives glomerulosclerosis and the progression of FSGS. PODCAST: This article contains a podcast at.

Stability of asymmetric cell division: A deformable cell model of cytokinesis applied to C. elegans.

Cell division during early embryogenesis is linked to key morphogenic events such as embryo symmetry breaking and tissue patterning. It is thought that the physical surrounding of cells together with cell intrinsic cues act as a mechanical "mold," guiding cell division to ensure these events are robust. To quantify how cell division is affected by the mechanical and geometrical environment, we present a novel computational mechanical model of cytokinesis, the final phase of cell division. Simulations with the model reproduced experimentally observed furrow dynamics and describe the volume ratio of daughter cells in asymmetric cell divisions, based on the position and orientation of the mitotic spindle. For dividing cells in geometrically confined environments, we show how the orientation of confinement relative to the division axis modulates the volume ratio in asymmetric cell division. Further, we quantified how cortex viscosity and surface tension determine the shape of a dividing cell and govern bubble-instabilities in asymmetric cell division. Finally, we simulated the formation of the three body axes via sequential (a)symmetric divisions up until the six-cell stage of early C. elegans development, which proceeds within the confines of an eggshell. We demonstrate how model input parameters spindle position and orientation provide sufficient information to reliably predict the volume ratio of daughter cells during the cleavage phase of development. However, for egg geometries perturbed by compression, the model predicts that a change in confinement alone is insufficient to explain experimentally observed differences in cell volume. This points to an effect of the compression on the spindle positioning mechanism. Additionally, the model predicts that confinement stabilizes asymmetric cell divisions against bubble-instabilities.

Convolutional Neural Networks for the Evaluation of Chronic and Inflammatory Lesions in Kidney Transplant Biopsies.

In kidney transplant biopsies, both inflammation and chronic changes are important features that predict long-term graft survival. Quantitative scoring of these features is important for transplant diagnostics and kidney research. However, visual scoring is poorly reproducible and labor intensive. The goal of this study was to investigate the potential of convolutional neural networks (CNNs) to quantify inflammation and chronic features in kidney transplant biopsies. A structure segmentation CNN and a lymphocyte detection CNN were applied on 125 whole-slide image pairs of periodic acid-Schiff- and CD3-stained slides. The CNN results were used to quantify healthy and sclerotic glomeruli, interstitial fibrosis, tubular atrophy, and inflammation within both nonatrophic and atrophic tubuli, and in areas of interstitial fibrosis. The computed tissue features showed high correlation with Banff lesion scores of five pathologists (A.A., A.Dend., J.H.B., J.K., and T.N.). Analyses on a small subset showed a moderate correlation toward higher CD3+ cell density within scarred regions and higher CD3+ cell count inside atrophic tubuli correlated with long-term change of estimated glomerular filtration rate. The presented CNNs are valid tools to yield objective quantitative information on glomeruli number, fibrotic tissue, and inflammation within scarred and non-scarred kidney parenchyma in a reproducible manner. CNNs have the potential to improve kidney transplant diagnostics and will benefit the community as a novel method to generate surrogate end points for large-scale clinical studies.

spheresDT/Mpacts-PiCS: cell tracking and shape retrieval in membrane-labeled embryos.

MOTIVATION: Uncovering the cellular and mechanical processes that drive embryo formation requires an accurate read out of cell geometries over time. However, automated extraction of 3D cell shapes from time-lapse microscopy remains challenging, especially when only membranes are labeled. RESULTS: We present an image analysis framework for automated tracking and three-dimensional cell segmentation in confocal time lapses. A sphere clustering approach allows for local thresholding and application of logical rules to facilitate tracking and unseeded segmentation of variable cell shapes. Next, the segmentation is refined by a discrete element method simulation where cell shapes are constrained by a biomechanical cell shape model. We apply the framework on Caenorhabditis elegans embryos in various stages of early development and analyze the geometry of the 7- and 8-cell stage embryo, looking at volume, contact area and shape over time. AVAILABILITY AND IMPLEMENTATION: The Python code for the algorithm and for measuring performance, along with all data needed to recreate the results is freely available at 10.5281/zenodo.5108416 and 10.5281/zenodo.4540092. The most recent version of the software is maintained at https://bitbucket.org/pgmsembryogenesis/sdt-pics. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The podocyte as a direct target of glucocorticoids in nephrotic syndrome.

Nephrotic syndrome (NS) is characterized by massive proteinuria; podocyte loss or altered function is a central event in its pathophysiology. Treatment with glucocorticoids is the mainstay of therapy, however, many patients experience one or multiple relapses and prolonged use may be associated with severe adverse effects. Recently the beneficial effects of glucocorticoids have been attributed to a direct effect on podocytes in addition to the well-known immunosuppressive effects. The molecular effects of glucocorticoid action have been studied using animal and cell models of NS. This review provides a comprehensive overview of different molecular mediators regulated by glucocorticoids, including an overview of the model systems that were used to study them. Glucocorticoids are described to stimulate podocyte recovery by restoring pro-survival signalling of slit diaphragm-related proteins and limiting inflammatory responses. Of special interest is the effect of glucocorticoids on stabilizing the cytoskeleton of podocytes, since these effects are also described for other therapeutic agents used in NS, such as cyclosporin. Current models provide much insight but do not fully recapitulate the human condition since the pathophysiology underlying NS is poorly understood. New and promising models include the glomerulus-on-a-chip and kidney organoids, which have the potential to be further developed into functional NS models in the future.

Establishment and characterization of a novel conditionally immortalized human parietal epithelial cell line.

Parietal epithelial cells (PECs) are epithelial cells in the kidney, surrounding Bowman's space. When activated, PECs increase in cell volume, proliferate, migrate to the glomerular tuft and excrete extracellular matrix. Activated PECs are crucially involved in the formation of sclerotic lesions, seen in focal segmental glomerulosclerosis (FSGS). In FSGS, a number of glomeruli show segmental sclerotic lesions. Further disease progression will lead to increasing number of involved glomeruli and gradual destruction of the affected glomeruli. Although the involvement of PECs in FSGS has been acknowledged, little is known about the molecular processes driving PEC activation. To get more insights in this process, accurate in vivo and in vitro models are needed. Here, we describe the development and characterization of a novel conditionally immortalized human PEC (ciPEC) line. We demonstrated that ciPECs are differentiated when grown under growth-restrictive conditions and express important PEC-specific markers, while lacking podocyte and endothelial markers. In addition, ciPECs showed PEC-like morphology and responded to IL-1ß treatment. We therefore conclude that we have successfully generated a novel PEC line, which can be used for future studies on the role of PECs in FSGS.

Investigating the Molecular Mechanisms of Renal Hepcidin Induction and Protection upon Hemoglobin-Induced Acute Kidney Injury.

Hemolysis is known to cause acute kidney injury (AKI). The iron regulatory hormone hepcidin, produced by renal distal tubules, is suggested to exert a renoprotective role during this pathology. We aimed to elucidate the molecular mechanisms of renal hepcidin synthesis and its protection against hemoglobin-induced AKI. In contrast to known hepatic hepcidin induction, incubation of mouse cortical collecting duct (mCCDcl1) cells with IL-6 or LPS did not induce Hamp1 mRNA expression, whereas iron (FeS) and hemin significantly induced hepcidin synthesis (p < 0.05). Moreover, iron/heme-mediated hepcidin induction in mCCDcl1 cells was caused by the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, as indicated by increased nuclear Nrf2 translocation and induced expression of Nrf2 downstream targets GCLM (p < 0.001), NQO1 (p < 0.001), and TXNRD1 (p < 0.005), which could be prevented by the known Nrf2 inhibitor trigonelline. Newly created inducible kidney-specific hepcidin KO mice demonstrated a significant reduction in renal Hamp1 mRNA expression. Phenylhydrazine (PHZ)-induced hemolysis caused renal iron loading and oxidative stress in both wildtype (Wt) and KO mice. PHZ treatment in Wt induced inflammatory markers (IL-6, TNFα) but not Hamp1. However, since PHZ treatment also significantly reduced systemic hepcidin levels in both Wt and KO mice (both p < 0.001), a dissection between the roles of systemic and renal hepcidin could not be made. Combined, the results of our study indicate that there are kidney-specific mechanisms in hepcidin regulation, as indicated by the dominant role of iron and not inflammation as an inducer of renal hepcidin, but also emphasize the complex interplay of various iron regulatory mechanisms during AKI on a local and systemic level.

Quantitative assessment of inflammatory infiltrates in kidney transplant biopsies using multiplex tyramide signal amplification and deep learning.

Delayed graft function (DGF) is a strong risk factor for development of interstitial fibrosis and tubular atrophy (IFTA) in kidney transplants. Quantitative assessment of inflammatory infiltrates in kidney biopsies of DGF patients can reveal predictive markers for IFTA development. In this study, we combined multiplex tyramide signal amplification (mTSA) and convolutional neural networks (CNNs) to assess the inflammatory microenvironment in kidney biopsies of DGF patients (n = 22) taken at 6 weeks post-transplantation. Patients were stratified for IFTA development (<10% versus ≥10%) from 6 weeks to 6 months post-transplantation, based on histopathological assessment by three kidney pathologists. One mTSA panel was developed for visualization of capillaries, T- and B-lymphocytes and macrophages and a second mTSA panel for T-helper cell and macrophage subsets. The slides were multi spectrally imaged and custom-made python scripts enabled conversion to artificial brightfield whole-slide images (WSI). We used an existing CNN for the detection of lymphocytes with cytoplasmatic staining patterns in immunohistochemistry and developed two new CNNs for the detection of macrophages and nuclear-stained lymphocytes. F1-scores were 0.77 (nuclear-stained lymphocytes), 0.81 (cytoplasmatic-stained lymphocytes), and 0.82 (macrophages) on a test set of artificial brightfield WSI. The CNNs were used to detect inflammatory cells, after which we assessed the peritubular capillary extent, cell density, cell ratios, and cell distance in the two patient groups. In this cohort, distance of macrophages to other immune cells and peritubular capillary extent did not vary significantly at 6 weeks post-transplantation between patient groups. CD163+ cell density was higher in patients with ≥10% IFTA development 6 months post-transplantation (p < 0.05). CD3+CD8-/CD3+CD8+ ratios were higher in patients with <10% IFTA development (p < 0.05). We observed a high correlation between CD163+ and CD4+GATA3+ cell density (R = 0.74, p < 0.001). Our study demonstrates that CNNs can be used to leverage reliable, quantitative results from mTSA-stained, multi spectrally imaged slides of kidney transplant biopsies.

The role of actin protrusion dynamics in cell migration through a degradable viscoelastic extracellular matrix: Insights from a computational model.

Actin protrusion dynamics plays an important role in the regulation of three-dimensional (3D) cell migration. Cells form protrusions that adhere to the surrounding extracellular matrix (ECM), mechanically probe the ECM and contract in order to displace the cell body. This results in cell migration that can be directed by the mechanical anisotropy of the ECM. However, the subcellular processes that regulate protrusion dynamics in 3D cell migration are difficult to investigate experimentally and therefore not well understood. Here, we present a computational model of cell migration through a degradable viscoelastic ECM. This model is a 2D representation of 3D cell migration. The cell is modeled as an active deformable object that captures the viscoelastic behavior of the actin cortex and the subcellular processes underlying 3D cell migration. The ECM is regarded as a viscoelastic material, with or without anisotropy due to fibrillar strain stiffening, and modeled by means of the meshless Lagrangian smoothed particle hydrodynamics (SPH) method. ECM degradation is captured by local fluidization of the material and permits cell migration through the ECM. We demonstrate that changes in ECM stiffness and cell strength affect cell migration and are accompanied by changes in number, lifetime and length of protrusions. Interestingly, directly changing the total protrusion number or the average lifetime or length of protrusions does not affect cell migration. A stochastic variability in protrusion lifetime proves to be enough to explain differences in cell migration velocity. Force-dependent adhesion disassembly does not result in faster migration, but can make migration more efficient. We also demonstrate that when a number of simultaneous protrusions is enforced, the optimal number of simultaneous protrusions is one or two, depending on ECM anisotropy. Together, the model provides non-trivial new insights in the role of protrusions in 3D cell migration and can be a valuable contribution to increase the understanding of 3D cell migration mechanics.

Distribution and propagation of mechanical stress in simulated structurally heterogeneous tissue spheroids.

The mechanical microenvironment of cells has been associated with phenotypic changes that cells undergo in three-dimensional spheroid culture formats. Radial asymmetry in mechanical stress - with compression in the core and tension at the periphery - has been analyzed by representing tissue spheroids as homogeneous visco-elastic droplets under surface tension. However, the influence of the granular microstructure of tissue spheroids in the distribution of mechanical stress in tissue spheroids has not been accounted for in a generic manner. Here, we quantify the distribution and propagation of mechanical forces in structurally heterogeneous multicellular assemblies. For this, we perform numerical simulations of a deformable cell model, which represents cells as elastic, contractile shells surrounding a liquid incompressible cytoplasm, interacting by means of non-specific adhesion. Using this model, we show how cell-scale properties such as cortical stiffness, active tension and cell-cell adhesive tension influence the distribution of mechanical stress in simulated tissue spheroids. Next, we characterize the transition at the tissue-scale from a homogeneous liquid droplet to a heterogeneous packed granular assembly.

Intercellular Adhesion Stiffness Moderates Cell Decoupling as a Function of Substrate Stiffness.

The interplay between cell-cell and cell-substrate interactions is complex yet necessary for the formation and healthy functioning of tissues. The same mechanosensing mechanisms used by the cell to sense its extracellular matrix also play a role in intercellular interactions. We used the discrete element method to develop a computational model of a deformable cell that includes subcellular components responsible for mechanosensing. We modeled a three-dimensional cell pair on a patterned (two-dimensional) substrate, a simple laboratory setup to study intercellular interactions. We explicitly modeled focal adhesions and adherens junctions. These mechanosensing adhesions matured, becoming stabilized by force. We also modeled contractile stress fibers that bind the discrete adhesions. The mechanosensing fibers strengthened upon stalling. Traction exerted on the substrate was used to generate traction maps (along the cell-substrate interface). These simulated maps are compared to experimental maps obtained via traction force microscopy. The model recreates the dependence on substrate stiffness of the tractions' spatial distribution, contractile moment of the cell pair, intercellular force, and number of focal adhesions. It also recreates the phenomenon of cell decoupling, in which cells exert forces separately when substrate stiffness increases. More importantly, the model provides viable molecular explanations for decoupling: mechanosensing mechanisms are responsible for competition between different fiber-adhesion configurations present in the cell pair. The point at which an increasing substrate stiffness becomes as high as that of the cell-cell interface is the tipping point at which configurations that favor cell-substrate adhesion dominate over those favoring cell-cell adhesion. This competition is responsible for decoupling.

Renal phospholipidosis and impaired magnesium handling in high-fat-diet-fed mice.

Hypomagnesemia (blood Mg2+ concentration <0.7 mM) is a common electrolyte disorder in patients with type 2 diabetes (T2D), but the etiology remains largely unknown. In patients with T2D, reduced blood Mg2+ levels are associated with an increased decline in renal function, independent of glycemic control and hypertension. To study the underlying mechanism of this phenomenon, we investigated the renal effects of hypomagnesemia in high-fat-diet (HFD)-fed mice. In mice fed a low dietary Mg2+, the HFD resulted in severe hypomagnesemia within 4 wk. Renal or intestinal Mg2+ wasting was not observed after 16 wk on the diets. Despite the absence of urinary or fecal Mg2+ loss, the HFD induced a reduction in the mRNA expression transient receptor potential melastatin type 6 in both the kidney and colon. mRNA expression of distal convoluted tubule (DCT)-specific genes was down-regulated by the LowMg-HFD, indicating atrophy of the DCT. The low dietary Mg2+ resulted in severe HFD-induced proximal tubule phospholipidosis, which was absent in mice on a NormalMg-HFD. This was accompanied by albuminuria, moderate renal damage, and alterations in renal energy metabolism, including enhanced gluconeogenesis and cholesterol synthesis. In conclusion, this study shows that hypomagnesemia is a consequence of diet-induced obesity and insulin resistance. Moreover, hypomagnesemia induces major structural changes in the diabetic kidney, including proximal tubular phospholipidosis, providing a novel mechanism for the increased renal decline in patients with hypomagnesemic T2D.-Kurstjens, S., Smeets, B., Overmars-Bos, C., Dijkman, H. B., den Braanker, D. J. W., de Bel, T., Bindels, R. J. M., Tack, C. J. J., Hoenderop, J. G. J., de Baaij, J. H. F. Renal phospholipidosis and impaired magnesium handling in high-fat-diet-fed mice.

Mechanodegradation of Polymers: A Limiting Factor of Mechanochemical Activation in the Production of Amorphous Solid Dispersions by Cryomilling.

In this study, we report on the influence of mechanochemical activation on the chemical stability of amorphous solid dispersions made up of indomethacin and hydroxypropyl methyl cellulose (HPMC), poly(vinylpyrrolidone) (PVP), poly(vinylpyrrolidone vinylacetate) (PVPVA), or Soluplus. In agreement with our recently published work, all applied carriers were found to be prone to polymer degradation. Covalent bonds within the polymers were cleaved and mechanoradicals were generated. Furthermore, decomposition of indomethacin was also observed but occurred only in the presence of polymers. Hence, it is proposed that the generated mechanoradicals from the polymers are responsible for the chemical degradation of indomethacin. Our study also strongly suggests the existence of a critical polymer- and process-dependent molecular weight limit "M∞", below which only limited mechanodegradation takes place since the lower-molecular-weight polymer PVP K12PF had a less profound influence on the degradation of indomethacin in comparison to PVP K25.

Nephrotic syndrome in a dish: recent developments in modeling in vitro.

Nephrotic syndrome is a heterogeneous disease, and one of the most frequent glomerular disorders among children. Depending on the etiology, it may result in end-stage renal disease and the need for renal replacement therapy. A dysfunctional glomerular filtration barrier, comprising of endothelial cells, the glomerular basement membrane and podocytes, characterizes nephrotic syndrome. Podocytes are often the primary target cells in the pathogenesis, in which not only the podocyte function but also their crosstalk with other glomerular cell types can be disturbed due to a myriad of factors. The pathophysiology of nephrotic syndrome is highly complex and studying molecular mechanisms in vitro requires state-of-the-art cell-based models resembling the in vivo situation and preferably a fully functional glomerular filtration barrier. Current advances in stem cell biology and microfluidic platforms have heralded a new era of three-dimensional (3D) cultures that might have the potential to recapitulate the glomerular filtration barrier in vitro. Here, we highlight the molecular basis of nephrotic syndrome and discuss requirements to accurately study nephrotic syndrome in vitro, including an overview of specific podocyte markers, cutting-edge stem cell organoids, and the implementation of microfluidic platforms. The development of (patho) physiologically relevant glomerular models will accelerate the identification of molecular targets involved in nephrotic syndrome and may be the harbinger of a new era of therapeutic avenues.

Deep Learning-Based Histopathologic Assessment of Kidney Tissue.

BACKGROUND: The development of deep neural networks is facilitating more advanced digital analysis of histopathologic images. We trained a convolutional neural network for multiclass segmentation of digitized kidney tissue sections stained with periodic acid-Schiff (PAS). METHODS: We trained the network using multiclass annotations from 40 whole-slide images of stained kidney transplant biopsies and applied it to four independent data sets. We assessed multiclass segmentation performance by calculating Dice coefficients for ten tissue classes on ten transplant biopsies from the Radboud University Medical Center in Nijmegen, The Netherlands, and on ten transplant biopsies from an external center for validation. We also fully segmented 15 nephrectomy samples and calculated the network's glomerular detection rates and compared network-based measures with visually scored histologic components (Banff classification) in 82 kidney transplant biopsies. RESULTS: The weighted mean Dice coefficients of all classes were 0.80 and 0.84 in ten kidney transplant biopsies from the Radboud center and the external center, respectively. The best segmented class was "glomeruli" in both data sets (Dice coefficients, 0.95 and 0.94, respectively), followed by "tubuli combined" and "interstitium." The network detected 92.7% of all glomeruli in nephrectomy samples, with 10.4% false positives. In whole transplant biopsies, the mean intraclass correlation coefficient for glomerular counting performed by pathologists versus the network was 0.94. We found significant correlations between visually scored histologic components and network-based measures. CONCLUSIONS: This study presents the first convolutional neural network for multiclass segmentation of PAS-stained nephrectomy samples and transplant biopsies. Our network may have utility for quantitative studies involving kidney histopathology across centers and provide opportunities for deep learning applications in routine diagnostics.

The Effect of Cortical Elasticity and Active Tension on Cell Adhesion Mechanics.

We consider a cell as an elastic, contractile shell surrounding a liquid incompressible cytoplasm and with nonspecific adhesion. We perform numerical simulations of this model to study the mechanics of cell-cell separation. By variation of parameters, we are able to recover well-known limits of the Johnson-Kendall-Roberts theory, the Derjaguin-Muller-Toporov model, adhesive vesicles with surface tension (Brochard-Wyart and de Gennes derivation), and thin elastic shells. We further locate biological cells on this parameter space by comparison to existing experiments on S180 cells. Using this model, we show that mechanical parameters can be obtained that are consistent with both dual pipette aspiration and micropipette aspiration, a problem not successfully tackled so far. We estimate a cortex elastic modulus of Ec ≈ 15 kPa, an effective cortex thickness of tc ≈ 0.3 µm, and an active tension of γ ≈ 0.4 nN/µm. With these parameters, a Johnson-Kendall-Roberts-like scaling of the separation force is recovered. Finally, the change of contact radius with applied force in a pull-off experiment was investigated. For small forces, a scaling similar to both the Brochard-Wyart and de Gennes derivation and the Derjaguin-Muller-Toporov model is found.