Transglutaminase-mediated stiffening of the glomerular basement membrane mitigates pressure-induced reductions in molecular sieving coefficient by reducing compression.

Proteinuria, the presence of high molecular weight proteins in the urine, is a primary indicator of chronic kidney disease. Proteinuria results from increased molecular permeability of the glomerular filtration barrier combined with saturation or defects in tubular protein reabsorption. Any solute that passes into the glomerular filtrate traverses the glomerular endothelium, the glomerular basement membrane, and the podocyte slit diaphragm. Damage to any layer of the filter has reciprocal effects on other layers to increase glomerular permeability. The GBM is thought to act as a compressible ultrafilter that has increased molecular selectivity with increased pressure due to compression that reduced the porosity of the GBM with increased pressure. In multiple forms of chronic kidney disease, crosslinking enzymes are upregulated and may act to increase GBM stiffness. Here we show that enzymatically crosslinking porcine GBM with transglutaminase increases the stiffness of the GBM and mitigates pressure-dependent reductions in molecular sieving coefficient. This was modeled mathematically using a modified membrane transport model accounting for GBM compression. Changes in the mechanical properties of the GBM may contribute to proteinuria through pressure-dependent effects on GBM porosity.

Peroxidasin is required for full viability in development and for maintenance of tissue mechanics in adults.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

In Vitro Models to Evaluate Molecular Permeability of the Kidney Filtration Barrier.

The glomerular basement membrane (GBM) is an important component of the kidney filtration barrier. The ability to evaluate the molecular transport properties of the GBM and determining how changes in the structure, composition, and mechanical properties of the GBM regulate its size selective transport properties may provide additional insight into glomerular function. This chapter details a method for making in vitro models of the glomerular filtration barrier using animal-derived decellularized glomeruli. FITC-labelled Ficoll is used as a filtration probe to evaluate the molecular transport properties during passive diffusion and under applied pressure. These systems can serve as a platform to evaluate the molecular permeability of basement membrane systems using conditions that simulate normal or pathophysiological conditions.

Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Zinc finger protein 24-dependent transcription factor SOX9 up-regulation protects tubular epithelial cells during acute kidney injury.

Transcriptional profiling studies have identified several protective genes upregulated in tubular epithelial cells during acute kidney injury (AKI). Identifying upstream transcriptional regulators could lead to the development of therapeutic strategies augmenting the repair processes. SOX9 is a transcription factor controlling cell-fate during embryonic development and adult tissue homeostasis in multiple organs including the kidneys. SOX9 expression is low in adult kidneys; however, stress conditions can trigger its transcriptional upregulation in tubular epithelial cells. SOX9 plays a protective role during the early phase of AKI and facilitates repair during the recovery phase. To identify the upstream transcriptional regulators that drive SOX9 upregulation in tubular epithelial cells, we used an unbiased transcription factor screening approach. Preliminary screening and validation studies show that zinc finger protein 24 (ZFP24) governs SOX9 upregulation in tubular epithelial cells. ZFP24, a Cys2-His2 (C2H2) zinc finger protein, is essential for oligodendrocyte maturation and myelination; however, its role in the kidneys or in SOX9 regulation remains unknown. Here, we found that tubular epithelial ZFP24 gene ablation exacerbated ischemia, rhabdomyolysis, and cisplatin-associated AKI. Importantly, ZFP24 gene deletion resulted in suppression of SOX9 upregulation in injured tubular epithelial cells. Chromatin immunoprecipitation and promoter luciferase assays confirmed that ZFP24 bound to a specific site in both murine and human SOX9 promoters. Importantly, CRISPR/Cas9-mediated mutation in the ZFP24 binding site in the SOX9 promoter in vivo led to suppression of SOX9 upregulation during AKI. Thus, our findings identify ZFP24 as a critical stress-responsive transcription factor protecting tubular epithelial cells through SOX9 upregulation.

A kidney proximal tubule model to evaluate effects of basement membrane stiffening on renal tubular epithelial cells.

The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell-matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell-cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell-cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.

Diabetic hyperglycemia promotes primary tumor progression through glycation-induced tumor extracellular matrix stiffening.

Diabetes mellitus is a complex metabolic disorder that is associated with an increased risk of breast cancer. Despite this correlation, the interplay between tumor progression and diabetes, particularly with regard to stiffening of the extracellular matrix, is still mechanistically unclear. Here, we established a murine model where hyperglycemia was induced before breast tumor development. Using the murine model, in vitro systems, and patient samples, we show that hyperglycemia increases tumor growth, extracellular matrix stiffness, glycation, and epithelial-mesenchymal transition of tumor cells. Upon inhibition of glycation or mechanotransduction in diabetic mice, these same metrics are reduced to levels comparable with nondiabetic tumors. Together, our study describes a novel biomechanical mechanism by which diabetic hyperglycemia promotes breast tumor progression via glycating the extracellular matrix. In addition, our work provides evidence that glycation inhibition is a potential adjuvant therapy for diabetic cancer patients due to the key role of matrix stiffening in both diseases.

Kidney-on-a-Chip: Mechanical Stimulation and Sensor Integration.

Bioengineered in vitro models of the kidney offer unprecedented opportunities to better mimic the in vivo microenvironment. Kidney-on-a-chip technology reproduces 2D or 3D features which can replicate features of the tissue architecture, composition, and dynamic mechanical forces experienced by cells in vivo. Kidney cells are exposed to mechanical stimuli such as substrate stiffness, shear stress, compression, and stretch, which regulate multiple cellular functions. Incorporating mechanical stimuli in kidney-on-a-chip is critically important for recapitulating the physiological or pathological microenvironment. This review will explore approaches to applying mechanical stimuli to different cell types using kidney-on-a-chip models and how these systems are used to study kidney physiology, model disease, and screen for drug toxicity. We further discuss sensor integration into kidney-on-a-chip for monitoring cellular responses to mechanical or other pathological stimuli. We discuss the advantages, limitations, and challenges associated with incorporating mechanical stimuli in kidney-on-a-chip models for a variety of applications. Overall, this review aims to highlight the importance of mechanical stimuli and sensor integration in the design and implementation of kidney-on-a-chip devices.

A Biomimetic In Vitro Model of the Kidney Filtration Barrier Using Tissue-Derived Glomerular Basement Membrane.

The glomerular filtration barrier (GFB) filters the blood to remove toxins while retaining high molecular weight proteins in the circulation. The glomerular basement membrane (GBM) and podocytes, highly specialized epithelial cells, are critical components of the filtration barrier. The GBM serves as a physical barrier to passage of molecules into the filtrate. Podocytes adhere to the filtrate side of the GBM and further restrict passage of high molecular weight molecules into the filtrate. Here, a 3D cell culture model of the glomerular filtration barrier to evaluate the role of the GBM and podocytes in mediating molecular diffusion is developed. GBM is isolated from mammalian kidneys to recapitulate the composition and mechanics of the in vivo basement membrane. The GFB model exhibits molecular selectivity that is comparable to the in vivo filtration barrier. The GBM alone provides a stringent barrier to passage of albumin and Ficoll. Podocytes further restrict molecular diffusion. Damage to the GBM that is typical of diabetic kidney disease is simulated using hypochlorous acid and results in increased molecular diffusion. This system can serve as a platform to evaluate the effects of GBM damage, podocyte injury, and reciprocal effects of altered podocyte-GBM interactions on kidney microvascular permeability.

Mechanical characterization of native and sugar-modified decellularized kidneys.

Decellularized organs have the potential to be used as scaffolds for tissue engineering organ replacements. The mechanical properties of the extracellular matrix (ECM) following decellularization are critical for structural integrity and for regulation of cell function upon recellularization. Advanced glycation end products (AGEs) accumulate in the ECM with age and their formation is accelerated by several pathological conditions including diabetes. Some AGEs span multiple amino acids to form crosslinks that may alter the mechanical properties of the ECM. The goal of this work was to evaluate how sugar-induced modifications to the ECM affect the mechanical behavior of decellularized kidney. The compressive and tensile properties of the kidney ECM were evaluated using an accelerated model of AGE formation by ribose. Results show that ribose modifications significantly alter the mechanical behavior of decellularized kidney. Increased resistance to deformation corresponds to increased ECM crosslinking, and mechanical changes can be partially mitigated by AGE inhibition. The degree of post-translational modification of the ECM is dependent on the age and health of the organ donor and may play a role in regulating the mechanical properties of decellularized organs.

Glycation alters the mechanical behavior of kidney extracellular matrix.

The mechanical properties of the extracellular matrix (ECM) are important in maintaining normal physiological function, and changes in ECM mechanics drive disease. The biochemical structure of the ECM is modified with aging and in diseases such as diabetes. One mechanism of ECM modification is the non-enzymatic reaction between sugars and ECM proteins resulting in formation of advanced glycation end products (AGEs). Some AGE reactions result in formation of molecular crosslinks within or between matrix proteins, but it is not clear how sugar-mediated biochemical modification of the ECM translates to changes in kidney ECM mechanical properties. AGE-mediated changes in ECM mechanics may have pathological consequences in diabetic kidney disease. To determine how sugars alter the mechanical properties of the kidney ECM, we employ custom methodologies to evaluate the mechanical properties of isolated tubular basement membrane (TBM) and glomerular ECM. Results show that the mechanical properties of TBM and glomerular ECM stiffness were altered by incubation in glucose and ribose. Mechanical behavior of TBM and glomerular ECM were further evaluated using mechanical models for hyperelastic materials in tension and compression. Increased ECM stiffness following sugar modification corresponded to increased crosslinking as determined by ECM fluorescence and reduced pepsin extractability of sugar modified ECM. These results show that sugar-induced modifications significantly affect the mechanical properties of kidney ECM. AGE-mediated changes in ECM mechanics may be important in progression of chronic diseases including diabetic kidney disease.

Application of physiological shear stress to renal tubular epithelial cells.

Renal tubular epithelial cells are consistently exposed to flow of glomerular filtrate that creates fluid shear stress at the apical cell surface. This biophysical stimulus regulates several critical renal epithelial cell functions, including transport, protein uptake, and barrier function. Defining the in vivo mechanical conditions in the kidney tubule is important for accurately recapitulating these conditions in vitro. Here we provide a summary of the fluid flow conditions in the kidney and how this translates into different levels of fluid shear stress down the length of the nephron. A detailed method is provided for measuring fluid flow in the proximal tubule by intravital microscopy. Devices to mimic in vivo fluid shear stress for in vitro studies are discussed, and we present two methods for culture and analysis of renal tubule epithelial cells exposed physiological levels of fluid shear stress. The first is a microfluidic device that permits application of controlled shear stress to cells cultured on porous membranes. The second is culture of renal tubule cells on an orbital shaker. Each method has advantages and disadvantages that should be considered in the context of the specific experimental objectives.

DSS-induced damage to basement membranes is repaired by matrix replacement and crosslinking.

Basement membranes are an ancient form of animal extracellular matrix. As important structural and functional components of tissues, basement membranes are subject to environmental damage and must be repaired while maintaining functions. Little is known about how basement membranes get repaired. This paucity stems from a lack of suitable in vivo models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult Drosophila DSS becomes incorporated into the basement membrane, promoting its expansion while decreasing its stiffness, which causes morphological changes to the underlying muscles. Remarkably, two days after withdrawal of DSS, the basement membrane is repaired by all measures of analysis. We used this new damage model to determine that repair requires collagen crosslinking and replacement of damaged components. Genetic and biochemical evidence indicates that crosslinking is required to stabilize the newly incorporated repaired Collagen IV rather than to stabilize the damaged Collagen IV. These results suggest that basement membranes are surprisingly dynamic.

Apical Shear Stress Enhanced Organic Cation Transport in Human OCT2/MATE1-Transfected Madin-Darby Canine Kidney Cells Involves Ciliary Sensing.

Active transport by renal proximal tubules plays a significant role in drug disposition. During drug development, estimates of renal excretion are essential to dose determination. Kidney bioreactors that reproduce physiologic cues in the kidney, such as flow-induced shear stress, may better predict in vivo drug behavior than do current in vitro models. In this study, we investigated the role of shear stress on active transport of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) by Madin-Darby canine kidney cells exogenously expressing the human organic cation transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1). Cells cultured in a parallel plate under continuous media perfusion formed a tight monolayer with a high barrier to inulin. In response to increasing levels of shear stress (0.2-2 dynes/cm2), cells showed a corresponding increase in transport of ASP+, reaching a maximal 4.2-fold increase at 2 dynes/cm2 compared with cells cultured under static conditions. This transport was inhibited with imipramine, indicating active transport was present under shear stress conditions. Cells exposed to shear stress of 2 dynes/cm2 also showed an increase in RNA expression of both transfected human and endogenous OCT2 (3.7- and 2.0-fold, respectively). Removal of cilia by ammonium sulfate eliminated the effects of shear on ASP+ transport at 0.5 dynes/cm2 with no effect on ASP+ transport under static conditions. These results indicate that shear stress affects active transport of organic cations in renal tubular epithelial cells in a cilia-dependent manner.

Substrate Elasticity Governs Differentiation of Renal Tubule Cells in Prolonged Culture.

IMPACT STATEMENT: Successful clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal proximal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this article, we show for the first time that in vitro substrate mechanical properties dictate differentiation of cultured renal proximal tubule cells. Remarkably, this effect was only discernable after 4 weeks in culture, longer than usually reported for this cell type. These results demonstrate a new tunable parameter to optimize cell differentiation in renal tissue engineering.

Orbital Shear Stress Regulates Differentiation and Barrier Function of Primary Renal Tubular Epithelial Cells.

Primary cells cultured in vitro gradually lose features characteristic of the in vivo phenotype. Culture techniques that help maintain cell-specific phenotype are advantageous for development of tissue engineered and bioartificial organs. Here we evaluated the phenotype of primary human renal tubular epithelial cells subjected to fluid shear stress by culturing the cells on an orbital shaker. Transepithelial electrical resistance (TEER), cell density, and gene and protein expression of proximal tubule-specific functional markers were measured in cells subjected to orbital shear stress. Cells cultured on an orbital shaker had increased TEER, higher cell density, and enhanced tubular epithelial specific gene and protein expression. This is likely due at least in part to the mechanical stress applied to the apical surface of the cells although other factors including increased nutrient and oxygen delivery and improved mixing could also play a role. These results suggest that orbital shaker culture may be a simple approach to augmenting the differentiated phenotype of cultured renal epithelial cells.

The sulfilimine cross-link of collagen IV contributes to kidney tubular basement membrane stiffness.

Basement membranes (BMs), a specialized form of extracellular matrix, underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of the BM influence cell-BM interactions. Collagen IV is a major constituent of the BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability, as observed with glomerular BM in Alport syndrome. These findings have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S = N) between a methionine sulfur and a lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. In peroxidasin knockout mice with reduced collagen IV sulfilimine cross-links, we find a reduction in renal tubular BM stiffness. Thus this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.

A modular microfluidic bioreactor with improved throughput for evaluation of polarized renal epithelial cells.

Most current microfluidic cell culture systems are integrated single use devices. This can limit throughput and experimental design options, particularly for epithelial cells, which require significant time in culture to obtain a fully differentiated phenotype. In addition, epithelial cells require a porous growth substrate in order to fully polarize their distinct apical and basolateral membranes. We have developed a modular microfluidic system using commercially available porous culture inserts to evaluate polarized epithelial cells under physiologically relevant fluid flow conditions. The cell-support for the bioreactor is a commercially available microporous membrane that is ready to use in a 6-well format, allowing for cells to be seeded in advance in replicates and evaluated for polarization and barrier function prior to experimentation. The reusable modular system can be easily assembled and disassembled using these mature cells, thus improving experimental throughput and minimizing fabrication requirements. The bioreactor consists of an apical microfluidic flow path and a static basolateral chamber that is easily accessible from the outside of the device. The basolateral chamber acts as a reservoir for transport across the cell layer. We evaluated the effect of initiation of apical shear flow on short-term intracellular signaling and mRNA expression using primary human renal epithelial cells (HRECs). Ten min and 5 h after initiation of apical fluid flow over a stable monolayer of HRECs, cells demonstrated increased phosphorylation of extracellular signal-related kinase and increased expression of interleukin 6 (IL-6) mRNA, respectively. This bioreactor design provides a modular platform with rapid experimental turn-around time to study various epithelial cell functions under physiologically meaningful flow conditions.

Shear stress is normalized in glomerular capillaries following ⠚ nephrectomy.

Loss of significant functional renal mass results in compensatory structural and hemodynamic adaptations in the nephron. While these changes have been characterized in several injury models, how they affect hemodynamic forces at the glomerular capillary wall has not been adequately characterized, despite their potential physiological significance. Therefore, we used intravital multiphoton microscopy to measure the velocity of red blood cells in individual glomerular capillaries of normal rats and rats subjected to ⅚ nephrectomy. Glomerular capillary blood flow rate and wall shear stress were then estimated using previously established experimental and mathematical models to account for changes in hematocrit and blood rheology in small vessels. We found little change in the hemodynamic parameters in glomerular capillaries immediately following injury. At 2 wk postnephrectomy, significant changes in individual capillary blood flow velocity and volume flow rate were present. Despite these changes, estimated capillary wall shear stress was unchanged. This was a result of an increase in capillary diameter and changes in capillary blood rheology in nephrectomized rats.

Large-scale proteomic characterization of melanoma expressed proteins reveals nestin and vimentin as biomarkers that can potentially distinguish melanoma subtypes.

Melanoma is an aggressive type of skin cancer, which accounts for only 4% of skin cancer cases but causes around 75% of skin cancer deaths. Currently, there is a limited set of protein biomarkers that can distinguish melanoma subtypes and provide an accurate prognosis of melanoma. Thus, we have selected and profiled the proteomes of five different melanoma cell lines from different stages of progression in comparison with a normal melanocytes using tandem mass spectrometry. We also profiled the proteome of a solid metastatic melanoma tumor. This resulted in the identification of 4758 unique proteins, among which ∼200-300 differentially expressed proteins from each set were found by quantitative proteomics. Correlating protein expression with aggressiveness of each melanoma cell line and literature mining resulted in the final selection of six proteins: vimentin, nestin, fibronectin, annexin A1, dipeptidyl peptidase IV, and histone H2A1B. Validation of nestin and vimentin using 40 melanoma samples revealed pattern of protein expression can help predict melanoma aggressiveness in different subgroups of melanoma. These results, together with the combined list of 4758 expressed proteins, provide a valuable resource for selecting melanoma biomarkers in the future for the clinical and research community.