Open-Source System for Real-Time Functional Assessment of In Vitro Filtration Barriers.

The integrity of the barrier between blood and the selective filtrate of solutes is important for homeostasis and its disruption contributes to many diseases. Microphysiological systems that incorporate synthetic or natural membranes with human cells can mimic biological filtration barriers, such as the glomerular filtration barrier in the kidney, and they can readily be used to study cellular filtration processes as well as drug effects and interactions. We present an affordable, open-source platform for the real-time monitoring of functional filtration status in engineered microphysiological systems. Using readily available components, our assay can linearly detect real-time concentrations of two target molecules, FITC-labeled inulin and Texas Red-labeled human-serum albumin, within clinically relevant ranges, and it can be easily modified for different target molecules of varying sizes and tags. We demonstrate the platform's ability to determine the concentration of our target molecules automatically and consistently. We show through an acellular context that the platform enables real-time tracking of size-dependent diffusion with minimal fluid volume loss and without manual extraction of media, making it suitable for continuous operational monitoring of filtration status in microphysiological system applications. The platform's affordability and integrability with microphysiological systems make it ideal for many precision medicine applications, including evaluation of drug nephrotoxicity and other forms of drug discovery.

Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier.

BACKGROUND: Slit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive. METHODS: We systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches. RESULTS: We demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome-associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin. CONCLUSIONS: Key components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.

The glomerular filtration barrier: a structural target for novel kidney therapies.

Loss of normal kidney function affects more than 10% of the population and contributes to morbidity and mortality. Kidney diseases are currently treated with immunosuppressive agents, antihypertensives and diuretics with partial but limited success. Most kidney disease is characterized by breakdown of the glomerular filtration barrier (GFB). Specialized podocyte cells maintain the GFB, and structure-function experiments and studies of intercellular communication between the podocytes and other GFB cells, combined with advances from genetics and genomics, have laid the groundwork for a new generation of therapies that directly intervene at the GFB. These include inhibitors of apolipoprotein L1 (APOL1), short transient receptor potential channels (TRPCs), soluble fms-like tyrosine kinase 1 (sFLT1; also known as soluble vascular endothelial growth factor receptor 1), roundabout homologue 2 (ROBO2), endothelin receptor A, soluble urokinase plasminogen activator surface receptor (suPAR) and substrate intermediates for coenzyme Q10 (CoQ10). These molecular targets converge on two key components of GFB biology: mitochondrial function and the actin-myosin contractile machinery. This Review discusses therapies and developments focused on maintaining GFB integrity, and the emerging questions in this evolving field.

Reconsidering Garth Robinson: fluid flow and the glomerular filtration barrier.

PURPOSE OF REVIEW: The goal of this review is to present recent models of the filtration barrier that may suggest mechanism-based treatments for proteinuric renal disease. The vast majority of renal failure occurs in diseases of glomerular proteinuria. The physiology of the filtration barrier remains incompletely understood, preventing invention of mechanism-based therapies. Research is currently dominated by molecular biology approaches to the kidney instead of engineering-based filtration and transport models. RECENT FINDINGS: Reexamination of two older paradigms (basement membrane and slit diaphragm) and critical analysis of newer models may provide mechanistic insight to guide further research. We expand on our theory of podocyte-basement membrane mechanical interactions and speculate on mechanisms of action of the leading treatment for proteinuria, angiotensin blockade. SUMMARY: Treatment of proteinuria remains largely empiric and based on inhibition of the renin-angiotensin-aldosterone system, with additional benefit from statins and vitamin D. Improved definition of transport phenomena in the capillary wall may suggest rational design of new interventions.

A Technique for Studying Glomerular Filtration Integrity in the Zebrafish Pronephros.

With the advances in next-generation sequencing and rapid filtering of candidate variants in diseased patients, it has been increasingly important to develop translatable in vivo models to study genetic changes. This allows for functional validation of pathogenic mutations and establishes a system to understand the etiology of disease. Due to the ease of genetic manipulation and rapid ex utero development, the zebrafish has become a valuable resource to study important biological processes, including nephrogenesis. The development and function of the zebrafish pronephros are akin to that of mammals. As such, they offer a tractable model to study kidney disease, especially diabetic nephropathy. However, in order to study kidney dysfunction in zebrafish it is imperative that an appropriate readout is available. The appearance of macro-proteins in patient's urine is indicative of defective kidney function. In this technical chapter, we describe the in vivo use of fluorescently tagged dextrans of different molecular weights to reveal the integrity of the zebrafish glomerular filtration barrier.

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier.

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

Podocyte-actin dynamics in health and disease.

Genetic studies of hereditary forms of nephrotic syndrome have identified several proteins that are involved in regulating the permselective properties of the glomerular filtration system. Further extensive research has elucidated the complex molecular basis of the glomerular filtration barrier and clearly established the pivotal role of podocytes in the pathophysiology of glomerular diseases. Podocyte architecture is centred on focal adhesions and slit diaphragms - multiprotein signalling hubs that regulate cell morphology and function. A highly interconnected actin cytoskeleton enables podocytes to adapt in order to accommodate environmental changes and maintain an intact glomerular filtration barrier. Actin-based endocytosis has now emerged as a regulator of podocyte integrity, providing an impetus for understanding the precise mechanisms that underlie the steady-state control of focal adhesion and slit diaphragm components. This Review outlines the role of actin dynamics and endocytosis in podocyte biology, and discusses how molecular heterogeneity in glomerular disorders could be exploited to deliver more rational therapeutic interventions, paving the way for targeted medicine in nephrology.

Par3A is dispensable for the function of the glomerular filtration barrier of the kidney.

Polarity signaling through the atypical PKC (aPKC)-Par polarity complex is essential for the development and maintenance of the podocyte architecture and the function of the glomerular filtration barrier of the kidney. To study the contribution of Par3A in this complex, we generated a novel Pard3 podocyte-specific knockout mouse model by targeting exon 6 of the Pard3 gene. Genetic deletion of Pard3a did not impair renal function, neither at birth nor later in life. Even challenging the animals did not result in glomerular disease. Despite its well-established role in aPKC-mediated signaling, Par3A appears to be dispensable for the function of the glomerular filtration barrier. Moreover, its homolog Pard3b, and not Pard3a, is the dominant Par3 gene expressed in podocytes and found at the basis of the slit diaphragm, where it partially colocalizes with podocin. In conclusion, Par3A function is either dispensable for slit diaphragm integrity, or compensatory mechanisms and a high redundancy of the different polarity proteins, including Par3B, Lgl, or PALS1, maintain the function of the glomerular filtration barrier, even in the absence of Par3A.

Glomerular protein separation as a mechanism for powering renal concentrating processes.

Various models have been proposed to explain the urine concentrating mechanism in mammals, however uncertainty remains regarding the origin of the energy required for the production of concentrated urine. We propose a novel mechanism for concentrating urine. We postulate that the energy for the concentrating process is derived from the osmotic potentials generated by the separation of afferent blood into protein-rich efferent blood and protein-deplete filtrate. These two streams run in mutual juxtaposition along the length of the nephron and are thus suitably arranged to provide the osmotic potential to concentrate the urine. The proposed model is able to qualitatively explain the production of various urine concentrations under different clinical conditions. An approach to testing the feasibility of the hypothesis is proposed.

Progress and controversies in unraveling the glomerular filtration mechanism.

PURPOSE OF REVIEW: At first sight, the glomerular filter appears like a problem that should be easily solved. The majority of researchers view the filter like an impermeable wall perforated by specialized and size-selective pores (pore model). However, the fact that this model is in conflict with many of the experimental findings suggests that it may not yet be complete. RECENT FINDINGS: In the more recent electrokinetic model, we have proposed including electrical effects (streaming potentials). The present review investigates how this can provide a relatively simple mechanistic explanation for the great majority of the so far unexplained characteristics of the filter, for example why the filter never clogs. SUMMARY: Understanding how the glomerular filter functions is a prerequisite to investigate the pathogenesis of proteinuric glomerular diseases and the link between glomerular proteinuria and cardiovascular disease.

Novel in vivo techniques to visualize kidney anatomy and function.

Intravital imaging using multiphoton microscopy (MPM) has become an increasingly popular and widely used experimental technique in kidney research over the past few years. MPM allows deep optical sectioning of the intact, living kidney tissue with submicron resolution, which is unparalleled among intravital imaging approaches. MPM has solved a long-standing critical technical barrier in renal research to study several complex and inaccessible cell types and anatomical structures in vivo in their native environment. Comprehensive and quantitative kidney structure and function MPM studies helped our better understanding of the cellular and molecular mechanisms of the healthy and diseased kidney. This review summarizes recent in vivo MPM studies with a focus on the glomerulus and the filtration barrier, although select, glomerulus-related renal vascular and tubular functions are also mentioned. The latest applications of serial MPM of the same glomerulus in vivo, in the intact kidney over several days, during the progression of glomerular disease are discussed. This visual approach, in combination with genetically encoded fluorescent markers of cell lineage, has helped track the fate and function (e.g., cell calcium changes) of single podocytes during the development of glomerular pathologies, and provided visual proof for the highly dynamic, rather than static, nature of the glomerular environment. Future intravital imaging applications have the promise to further push the limits of optical microscopy, and to advance our understanding of the mechanisms of kidney injury. Also, MPM will help to study new mechanisms of tissue repair and regeneration, a cutting-edge area of kidney research.

Point: Proposing the electrokinetic model.

It is still not fully resolved how the glomerular filter works and why it never clogs. Several models have been proposed. In this review, we will compare the most widely used "pore model" to the more recent and refined "electrokinetic model" of glomerular filtration. The pore model assumes the existence of highly ordered regular pores, but it cannot provide a mechanistic explanation for several of the inherent characteristics of the glomerular filter. The electrokinetic model assumes that streaming potentials generate an electrical field along the filter surface which repels the negatively charged plasma proteins, preventing them from passing across the filter. The electrokinetic model can provide elegant mechanistic solutions for most of the unresolved riddles about the glomerular filter.

Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier.

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

Inhibition of the TRPC5 ion channel protects the kidney filter.

An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

Recent insights into the pathogenesis of nephrotic syndrome.

Nephrotic syndrome is characterized by heavy proteinuria followed by hypoproteinemia, hypercholestrolemia, lipiduria, and edema. The glomerular filtration barrier (GFB) consists of glomerular endothelial cells covered with glycocalyx, the basement membrane, subpodocyte space and podocytes with foot processes and slit membranes between them. The coordinated function of GFB has been considered to be the major barrier against filtration of plasma proteins to urine. However, new hypothesis suggesting more permeable GFB has emerged. According to this, proteinuria might be prevented by tubular protein reabsorbtion. Experiments and human studies have revealed numerous putative permeability factors in idiopathic nephrotic syndrome (minimal change disease/focal segmental glomerulosclerosis). New antigens and antibodies have been suggested in "idiopathic" membranous nephropathy as well. Formation of nephrotic edema, the role of oncotic pressure and of different sodium and water retaining hormones have been subject of intensive study. These findings should pave the way to new therapeutic modalities targeted more precisely to the pathogenic mechanisms.

Adiponectin promotes functional recovery after podocyte ablation.

Low levels of the adipocyte-secreted protein adiponectin correlate with albuminuria in both mice and humans, but whether adiponectin has a causative role in modulating renal disease is unknown. Here, we first generated a mouse model that allows induction of caspase-8-mediated apoptosis specifically in podocytes upon injection of a construct-specific agent. These POD-ATTAC mice exhibited significant kidney damage, mimicking aspects of human renal disease, such as foot process effacement, mesangial expansion, and glomerulosclerosis. After the initial induction, both podocytes and filtration function recovered. Next, we crossed POD-ATTAC mice with mice lacking or overexpressing adiponectin. POD-ATTAC mice lacking adiponectin developed irreversible albuminuria and renal failure; conversely, POD-ATTAC mice overexpressing adiponectin recovered more rapidly and exhibited less interstitial fibrosis. In conclusion, these results suggest that adiponectin is a renoprotective protein after podocyte injury. Furthermore, the POD-ATTAC mouse provides a platform for further studies, allowing precise timing of podocyte injury and regeneration.

Quantification of the electrostatic properties of the glomerular filtration barrier modeled as a charged fiber matrix separating anionic from neutral Ficoll.

In the current study we explore the electrostatic interactions on the transport of anionic Ficoll (aFicoll) vs. neutral Ficoll (nFicoll) over the glomerular filtration barrier (GFB) modeled as a charged fiber matrix. We first analyze experimental sieving data for the rat glomerulus, and second, we explore some of the basic implications of a theoretical model for the electrostatic interactions between a charged solute and a charged fiber-matrix barrier. To explain the measured difference in glomerular transport between nFicoll and aFicoll (Axelsson J, Sverrisson K, Rippe A, Fissell W, Rippe B. Am J Physiol 301: F708-F712, 2011), the present simulations demonstrate that the surface charge density needed on a charged fiber matrix must lie between -0.005 C/m(2) and -0.019 C/m(2), depending on the surface charge density of the solute. This is in good agreement with known surface charge densities for many proteins in the body. In conclusion, the current results suggest that electrical charge makes a moderate contribution to glomerular permeability, while molecular size and conformation seem to be more important. Yet, the weak electrical charge obtained in this study can be predicted to nearly totally exclude albumin from permeating through "high-selectivity" pathways in a charged-fiber matrix of the GFB.