The D2D3 form of uPAR acts as an immunotoxin and may cause diabetes and kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) is a risk factor for kidney diseases. In addition to suPAR, proteolysis of membrane-bound uPAR results in circulating D1 and D2D3 proteins. We showed that when exposed to a high-fat diet, transgenic mice expressing D2D3 protein developed progressive kidney disease marked by microalbuminuria, elevated serum creatinine, and glomerular hypertrophy. D2D3 transgenic mice also exhibited insulin-dependent diabetes mellitus evidenced by decreased levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, decreased pancreatic ß cell mass, and high fasting blood glucose. Injection of anti-uPAR antibody restored ß cell mass and function in D2D3 transgenic mice. At the cellular level, the D2D3 protein impaired ß cell proliferation and inhibited the bioenergetics of ß cells, leading to dysregulated cytoskeletal dynamics and subsequent impairment in the maturation and trafficking of insulin granules. D2D3 protein was predominantly detected in the sera of patients with nephropathy and insulin-dependent diabetes mellitus. These sera inhibited glucose-stimulated insulin release from human islets in a D2D3-dependent manner. Our study showed that D2D3 injures the kidney and pancreas and suggests that targeting this protein could provide a therapy for kidney diseases and insulin-dependent diabetes mellitus.

The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5.

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.

Podocyte-specific deletion of miR-146a increases podocyte injury and diabetic kidney disease.

Diabetic glomerular injury is a major complication of diabetes mellitus and is the leading cause of end stage renal disease (ESRD). Healthy podocytes are essential for glomerular function and health. Injury or loss of these cells results in increased proteinuria and kidney dysfunction and is a common finding in various glomerulopathies. Thus, mechanistic understanding of pathways that protect podocytes from damage are essential for development of future therapeutics. MicroRNA-146a (miR-146a) is a negative regulator of inflammation and is highly expressed in myeloid cells and podocytes. We previously reported that miR-146a levels are significantly reduced in the glomeruli of patients with diabetic nephropathy (DN). Here we report generation of mice with selective deletion of miR-146a in podocytes and use of these mice in models of glomerular injury. Induction of glomerular injury in C57BL/6 wildtype mice (WT) and podocyte-specific miR-146a knockout (Pod-miR146a-/-) animals via administration of low-dose lipopolysaccharide (LPS) or nephrotoxic serum (NTS) resulted in increased proteinuria in the knockout mice, suggesting that podocyte-expressed miR-146a protects these cells, and thus glomeruli, from damage. Furthermore, induction of hyperglycemia using streptozotocin (STZ) also resulted in an accelerated development of glomerulopathy and a rapid increase in proteinuria in the knockout animals, as compared to the WT animals, further confirming the protective role of podocyte-expressed miR-146a. We also confirmed that the direct miR-146a target, ErbB4, was significantly upregulated in the diseased glomeruli and erlotinib, an ErbB4 and EGFR inhibitor, reducedits upregulation and the proteinuria in treated animals. Primary miR146-/- podocytes from these animals also showed a basally upregulated TGFß-Smad3 signaling in vitro. Taken together, this study shows that podocyte-specific miR-146a is imperative for protecting podocytes from glomerular damage, via modulation of ErbB4/EGFR, TGFß, and linked downstream signaling.

Simultaneous stabilization of actin cytoskeleton in multiple nephron-specific cells protects the kidney from diverse injury.

Chronic kidney diseases and acute kidney injury are mechanistically distinct kidney diseases. While chronic kidney diseases are associated with podocyte injury, acute kidney injury affects renal tubular epithelial cells. Despite these differences, a cardinal feature of both acute and chronic kidney diseases is dysregulated actin cytoskeleton. We have shown that pharmacological activation of GTPase dynamin ameliorates podocyte injury in murine models of chronic kidney diseases by promoting actin polymerization. Here we establish dynamin's role in modulating stiffness and polarity of renal tubular epithelial cells by crosslinking actin filaments into branched networks. Activation of dynamin's crosslinking capability by a small molecule agonist stabilizes the actomyosin cortex of the apical membrane against injury, which in turn preserves renal function in various murine models of acute kidney injury. Notably, a dynamin agonist simultaneously attenuates podocyte and tubular injury in the genetic murine model of Alport syndrome. Our study provides evidence for the feasibility and highlights the benefits of novel holistic nephron-protective therapies.

Deiodinase-3 is a thyrostat to regulate podocyte homeostasis.

BACKGROUND: Nephrotic syndrome (NS) is associated with kidney podocyte injury and may occur as part of thyroid autoimmunity such as Graves' disease. Therefore, the present study was designed to ascertain if and how podocytes respond to and regulate the input of biologically active thyroid hormone (TH), 3,5,3'-triiodothyronine (T3); and also to decipher the pathophysiological role of type 3 deiodinase (D3), a membrane-bound selenoenzyme that inactivates TH, in kidney disease. METHODS: To study D3 function in healthy and injured (PAN, puromycin aminonucleoside and LPS, Lipopolysaccharide-mediated) podocytes, immunofluorescence, qPCR and podocyte-specific D3 knockout mouse were used. Surface plasmon resonance (SPR), co-immunoprecipitation and Proximity Ligation Assay (PLA) were used for the interaction studies. FINDINGS: Healthy podocytes expressed D3 as the predominant deiodinase isoform. Upon podocyte injury, levels of Dio3 transcript and D3 protein were dramatically reduced both in vitro and in the LPS mouse model of podocyte damage. D3 was no longer directed to the cell membrane, it accumulated in the Golgi and nucleus instead. Further, depleting D3 from the mouse podocytes resulted in foot process effacement and proteinuria. Treatment of mouse podocytes with T3 phenocopied the absence of D3 and elicited activation of αvß3 integrin signaling, which led to podocyte injury. We also confirmed presence of an active thyroid stimulating hormone receptor (TSH-R) on mouse podocytes, engagement and activation of which resulted in podocyte injury. INTERPRETATION: The study provided a mechanistic insight into how D3-αvß3 integrin interaction can minimize T3-dependent integrin activation, illustrating how D3 could act as a renoprotective thyrostat in podocytes. Further, injury caused by binding of TSH-R with TSH-R antibody, as found in patients with Graves' disease, explained a plausible link between thyroid disorder and NS. FUNDING: This work was supported by American Thyroid Association (ATA-2018-050.R1).

Renal cell markers: lighthouses for managing renal diseases.

Kidneys, one of the vital organs in our body, are responsible for maintaining whole body homeostasis. The complexity of renal function (e.g., filtration, reabsorption, fluid and electrolyte regulation, and urine production) demands diversity not only at the level of cell types but also in their overall distribution and structural framework within the kidney. To gain an in depth molecular-level understanding of the renal system, it is imperative to discern the components of kidney and the types of cells residing in each of the subregions. Recent developments in labeling, tracing, and imaging techniques have enabled us to mark, monitor, and identify these cells in vivo with high efficiency in a minimally invasive manner. In this review, we summarize different cell types, specific markers that are uniquely associated with those cell types, and their distribution in the kidney, which altogether make kidneys so special and different. Cellular sorting based on the presence of certain proteins on the cell surface allowed for the assignment of multiple markers for each cell type. However, different studies using different techniques have found contradictions in cell type-specific markers. Thus, the term "cell marker" might be imprecise and suboptimal, leading to uncertainty when interpreting the data. Therefore, we strongly believe that there is an unmet need to define the best cell markers for a cell type. Although the compendium of renal-selective marker proteins presented in this review is a resource that may be useful to researchers, we acknowledge that the list may not be necessarily exhaustive.

Metabolic Changes in Peripheral Blood Mononuclear Cells Isolated From Patients With End Stage Renal Disease.

As numerous complex pathologies stem from cellular energy dysfunction, we aimed to elucidate mitochondrial function and associated stress pathologies in kidney disease in a cohort of hemodialysis patients with end-stage kidney disease (ESKD). The bioenergetics study was conducted using peripheral blood mononuclear cells (PBMCs) of ESKD patients (n = 29) and healthy controls (no ESKD, n = 10). PBMCs were isolated from whole blood and seeded into assay plates to detect changes in oxidative phosphorylation and glycolysis. The bioenergetics analysis (i.e., mitochondrial stress test) was performed using Seahorse XFe24 flux analyzer. We observed significant reduction in mitochondrial respiration in patient PBMCs in terms of fundamental bioenergetics parameters such as basal respiration, ATP turnover, maximal respiration and spare respiratory capacity. These findings were correlated with the expression levels of proteins coordinating cellular energy status and regulating mitochondrial dynamics. Our data demonstrates an association between mitochondrial oxygen consumption of PBMCs and ESKD. AMPK activity, its downstream effector PGC-1α and mitochondrial fission/fusion proteins are partially responsible for the decrease in oxidative phosphorylation of PBMCs isolated from ESKD patients. We propose a link between mitochondrial dysfunction and ESKD and a role for mitochondria as a potential site for therapeutic interventions.

From Infancy to Fancy: A Glimpse into the Evolutionary Journey of Podocytes in Culture.

Podocytes are critical components of the filtration barrier and responsible for maintaining healthy kidney function. An assault on podocytes is generally associated with progression of chronic glomerular diseases. Therefore, podocyte pathophysiology is a favorite research subject for nephrologists. Despite this, podocyte research has lagged because of the unavailability of techniques for culturing such specialized cells ex vivo in quantities that are adequate for mechanistic studies. In recent years, this problem was circumvented by the efforts of researchers, who successfully developed several in vitro podocyte cell culture model systems that paved the way for incredible discoveries in the field of nephrology. This review sets us on a journey that provides a comprehensive insight into the groundbreaking breakthroughs and novel technologic advances made in the field of podocyte cell culture so far, beginning from its inception, evolution, and progression. In this study, we also describe in detail the pros and cons of different models that are being used to culture podocytes. Our extensive and exhaustive deliberation on the status of podocyte cell culture will facilitate researchers to choose wisely an appropriate model for their own research to avoid potential pitfalls in the future.

Soluble Urokinase Receptor and Acute Kidney Injury.

BACKGROUND: Acute kidney injury is common, with a major effect on morbidity and health care utilization. Soluble urokinase plasminogen activator receptor (suPAR) is a signaling glycoprotein thought to be involved in the pathogenesis of kidney disease. We investigated whether a high level of suPAR predisposed patients to acute kidney injury in multiple clinical contexts, and we used experimental models to identify mechanisms by which suPAR acts and to assess it as a therapeutic target. METHODS: We measured plasma levels of suPAR preprocedurally in patients who underwent coronary angiography and patients who underwent cardiac surgery and at the time of admission to the intensive care unit in critically ill patients. We assessed the risk of acute kidney injury at 7 days as the primary outcome and acute kidney injury or death at 90 days as a secondary outcome, according to quartile of suPAR level. In experimental studies, we used a monoclonal antibody to urokinase plasminogen activator receptor (uPAR) as a therapeutic strategy to attenuate acute kidney injury in transgenic mice receiving contrast material. We also assessed cellular bioenergetics and generation of reactive oxygen species in human kidney proximal tubular (HK-2) cells that were exposed to recombinant suPAR. RESULTS: The suPAR level was assessed in 3827 patients who were undergoing coronary angiography, 250 who were undergoing cardiac surgery, and 692 who were critically ill. Acute kidney injury developed in 318 patients (8%) who had undergone coronary angiography. The highest suPAR quartile (vs. the lowest) had an adjusted odds ratio of 2.66 (95% confidence interval [CI], 1.77 to 3.99) for acute kidney injury and 2.29 (95% CI, 1.71 to 3.06) for acute kidney injury or death at 90 days. Findings were similar in the surgical and critically ill cohorts. The suPAR-overexpressing mice that were given contrast material had greater functional and histologic evidence of acute kidney injury than wild-type mice. The suPAR-treated HK-2 cells showed heightened energetic demand and mitochondrial superoxide generation. Pretreatment with a uPAR monoclonal antibody attenuated kidney injury in suPAR-overexpressing mice and normalized bioenergetic changes in HK-2 cells. CONCLUSIONS: High suPAR levels were associated with acute kidney injury in various clinical and experimental contexts. (Funded by the National Institutes of Health and others.).

Nonimmune cell-derived ICOS ligand functions as a renoprotective αvß3 integrin-selective antagonist.

Soluble urokinase receptor (suPAR) is a circulatory molecule that activates αvß3 integrin on podocytes, causes foot process effacement, and contributes to proteinuric kidney disease. While active integrin can be targeted by antibodies and small molecules, endogenous inhibitors haven't been discovered yet. Here we report what we believe is a novel renoprotective role for the inducible costimulator ligand (ICOSL) in early kidney disease through its selective binding to podocyte αvß3 integrin. Contrary to ICOSL's immune-regulatory role, ICOSL in nonhematopoietic cells limited the activation of αvß3 integrin. Specifically, ICOSL contains the arginine-glycine-aspartate (RGD) motif, which allowed for a high-affinity and selective binding to αvß3 and modulation of podocyte adhesion. This binding was largely inhibited either by a synthetic RGD peptide or by a disrupted RGD sequence in ICOSL. ICOSL binding favored the active αvß3 rather than the inactive form and showed little affinity for other integrins. Consistent with the rapid induction of podocyte ICOSL by inflammatory stimuli, glomerular ICOSL expression was increased in biopsies of early-stage human proteinuric kidney diseases. Icosl deficiency in mice resulted in an increased susceptibility to proteinuria that was rescued by recombinant ICOSL. Our work identified a potentially novel role for ICOSL, which serves as an endogenous αvß3-selective antagonist to maintain glomerular filtration.

More expression, less function: cleaved dynamin in glomerular kidney disease.

The role of dynamin in regulation of kidney filtration barrier is well documented. Dynamin binds to and produces filamentous actin, which is a key component of healthy podocyte foot processes (FPs). Destruction of dynamin, for example by cathepsin L, leads to loss of a functional actin network and uncoordinated membrane signaling, a situation that allows for effacement of FPs and proteinuria. Now, Khalil et al have examined the dynamin expression in kidneys of proteinuric animal models as well as in kidney patients and produced data that further clarifies the role of dynamin in glomerular and tubular proteinuria and may aid in pinpointing patients who are affected by loss of dynamin function and may benefit from appropriate therapeutic approaches. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Podocytes: Way to Go.

This commentary highlights the article by Hara et al that discusses the clinical implications of mitotic catastrophe in podocyte health during diabetic kidney disease.

Single-nephron proteomes connect morphology and function in proteinuric kidney disease.

In diseases of many parenchymatous organs, heterogeneous deterioration of individual functional units determines the clinical prognosis. However, the molecular characterization at the level of such individual subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Proteinuric glomerular kidney diseases are frequent and assorted diseases affecting a fraction of glomeruli and their draining tubules to variable extents, and for which no specific treatment exists. Here, we developed and applied a mass spectrometry-based methodology to investigate heterogeneity of proteomes from individually isolated nephron segments from mice with proteinuric kidney disease. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins while genetic ablation of LAMP1-correlated lysosomal proteases could ameliorate glomerular damage in vivo. Furthermore, proteomic analyses of individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases revealed increased abundance of lysosomal proteins, in combination with a decreased abundance of mutated gene products. Thus, altered protein homeostasis (proteostasis) is a conserved key mechanism in proteinuric kidney diseases. Moreover, our technology can capture intra-individual variability in diseases of the kidney and other tissues at a sub-biopsy scale.

Podocytes exhibit a specialized protein quality control employing derlin-2 in kidney disease.

Podocytes are terminally differentiated cells of the kidney filtration barrier with a limited proliferative capacity and are the primary glomerular target for various sources of cellular stress. Accordingly, it is particularly important for podocytes to cope with stress efficiently to circumvent cell death and avoid compromising renal function. Improperly folded proteins within the endoplasmic reticulum (ER) are associated with increased cellular injury and cell death. To relieve ER stress, protein quality control mechanisms like ER-associated degradation (ERAD) are initiated. Derlin-2 is an important dislocation channel component in the ERAD pathway, having an indispensable role in clearing misfolded glycoproteins from the ER lumen. With studies linking ER stress to kidney disease, we investigated the role of derlin-2 in the susceptibility of podocytes to injury due to protein misfolding. We show that podocytes employ derlin-2 to mediate the ER quality control system to maintain cellular homeostasis in both mouse and human glomeruli. Patients with focal segmental glomerulosclerosis (FSGS) or diabetic nephropathy (DN) upregulate derlin-2 expression in response to glomerular injury, as do corresponding mouse models. In derlin-2-deficient podocytes, compensatory responses were lost under adriamycin (ADR)-induced ER dysfunction, and severe cellular injury ensued via a caspase-12-dependent pathway. Moreover, derlin-2 overexpression in vitro attenuated ADR-induced podocyte injury. Thus derlin-2 is part of a protein quality control mechanism that can rescue glomerular injury attributable to impaired protein folding pathways in the ER. Induction of derlin-2 expression in vivo may have applications in prevention and treatment of glomerular diseases.

TRPC5 Does Not Cause or Aggravate Glomerular Disease.

Transient receptor potential channel 5 (TRPC5) is highly expressed in brain and kidney and mediates calcium influx and promotes cell migration. In the kidney, loss of TRPC5 function has been reported to benefit kidney filter dynamics by balancing podocyte cytoskeletal remodeling. However, in vivo gain-in-function studies of TRPC5 with respect to kidney function have not been reported. To address this gap, we developed two transgenic mouse models on the C57BL/6 background by overexpressing either wild-type TRPC5 or a TRPC5 ion-pore mutant. Compared with nontransgenic controls, neither transgenic model exhibited an increase in proteinuria at 8 months of age or a difference in LPS-induced albuminuria. Moreover, activation of TRPC5 by Englerin A did not stimulate proteinuria, and inhibition of TRPC5 by ML204 did not significantly lower the level of LPS-induced proteinuria in any group. Collectively, these data suggest that the overexpression or activation of the TRPC5 ion channel does not cause kidney barrier injury or aggravate such injury under pathologic conditions.

High-content screening assay-based discovery of paullones as novel podocyte-protective agents.

Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. A podocyte's normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (F-actin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cell's filamentous network, is still lacking, partly because of unavailability of appropriate cellular assays for use in a drug discovery environment. Here, we describe a new high-content screening-based methodology and its implementation on podocytes to identify paullone derivatives as a novel group of podocyte-protective compounds. We find that three compounds, i.e., kenpaullone, 1-azakenpaullone, and alsterpaullone, dose dependently protect podocytes from puromycin aminonucleoside (PAN)-mediated injury in vitro by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3ß and p38 mitogen-activated protein kinase. In vivo it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone derivatives as novel podocyte-protective agents for future therapeutic development.

Soluble Urokinase Receptor and the Kidney Response in Diabetes Mellitus.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) worldwide. DN typically manifests by glomerular hyperfiltration and microalbuminuria; then, the disease progresses to impaired glomerular filtration rate, which leads to ESRD. Treatment options for DN include the strict control of blood glucose levels and pressure (e.g., intraglomerular hypertension). However, the search for novel therapeutic strategies is ongoing. These include seeking specific molecules that contribute to the development and progression of DN to potentially interfere with these "molecular targets" as well as with the cellular targets within the kidney such as podocytes, which play a major role in the pathogenesis of DN. Recently, podocyte membrane protein urokinase receptor (uPAR) and its circulating form (suPAR) are found to be significantly induced in glomeruli and sera of DN patients, respectively, and elevated suPAR levels predicted diabetic kidney disease years before the occurrence of microalbuminuria. The intent of this review is to summarize the emerging evidence of uPAR and suPAR in the clinical manifestations of DN. The identification of specific pathways that govern DN will help us build a more comprehensive molecular model for the pathogenesis of the disease that can inform new opportunities for treatment.

A tripartite complex of suPAR, APOL1 risk variants and αvß3 integrin on podocytes mediates chronic kidney disease.

Soluble urokinase plasminogen activator receptor (suPAR) independently predicts chronic kidney disease (CKD) incidence and progression. Apolipoprotein L1 (APOL1) gene variants G1 and G2, but not the reference allele (G0), are associated with an increased risk of CKD in individuals of recent African ancestry. Here we show in two large, unrelated cohorts that decline in kidney function associated with APOL1 risk variants was dependent on plasma suPAR levels: APOL1-related risk was attenuated in patients with lower suPAR, and strengthened in those with higher suPAR levels. Mechanistically, surface plasmon resonance studies identified high-affinity interactions between suPAR, APOL1 and αvß3 integrin, whereby APOL1 protein variants G1 and G2 exhibited higher affinity for suPAR-activated avb3 integrin than APOL1 G0. APOL1 G1 or G2 augments αvß3 integrin activation and causes proteinuria in mice in a suPAR-dependent manner. The synergy of circulating factor suPAR and APOL1 G1 or G2 on αvß3 integrin activation is a mechanism for CKD.

Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles.

Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.

A High-Content Screening Technology for Quantitatively Studying Podocyte Dynamics.

Podocytes form the visceral layer of a kidney glomerulus and express a characteristic octopus-like cellular architecture specialized for the ultrafiltration of blood. The cytoskeletal dynamics and structural elasticity of podocytes rely on the self-organization of highly interconnected actin bundles, and the maintenance of these features is important for the intact glomerular filtration. Development of more differentiated podocytes in culture has dramatically increased our understanding of the molecular mechanisms regulating podocyte actin dynamics. Podocytes are damaged in a variety of kidney diseases, and therapies targeting podocytes are being investigated with increasing efforts. Association between podocyte damage and disease severity-or between podocyte recovery and the performance of therapeutic molecules-have been the venues of research for years. In this perspective, more standardized high--content screening has emerged as a powerful tool for visualization and analysis of podocyte morphology. This high-throughput fluorescence microscopy technique is based on an automated image analysis with simultaneous detection of various phenotypes (multiplexing) across multiple phenotypic parameters (multiparametric). Here, we review the principles of high-content screening technology and summarize efforts to carry out small compound screen using podocytes.