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.

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.

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.

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.

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.

Regulation of G-protein signaling via Gnas is required to regulate proximal tubular growth in the Xenopus pronephros.

In the kidney, proximal tubules are very important for the reabsorption of water, ions and organic solutes from the primary urine. They are composed of highly specialized epithelial cells that are characterized by an elaborate apical brush border to increase transport efficiency. Using the pronephric kidney of Xenopus laevis we discovered that the G-protein modulator cholera toxin resulted in a dramatic reduction of the proximal tubular size. This phenotype was accompanied by changes in the cytoarchitecture characterized by ectopic expression of the distal tubular marker 4A6 and an impairment of yolk platelet degradation. In addition, cholera toxin caused edema formation. However, this phenotype was not due to kidney defects, but rather due to impaired vasculature development. Based on experiments with antisense morpholino oligomers as well as pharmacological agonists and antagonists, we could show that the complex phenotype of cholera toxin in the pronephric kidney was caused by the hyperactivation of a single G-protein alpha subunit, Gnas. This-in turn-caused elevated cAMP levels, triggered a Rapgef4-dependent signaling cassette and perturbed exo- and endocytosis. This perturbation of the secretory pathway by Ctx was not only observed in Xenopus embryos. Also, in a human proximal tubular cell line, cholera toxin or a Rapgef4-specific agonist increased uptake and decreased secretion of FITC-labeled Albumin. Based on these data we propose that the Gnas/cAMP/Rapgef4 pathway regulates the signals inducing the proliferation of proximal tubules to acquire their final organ size.

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.

Effects of pressure and electrical charge on macromolecular transport across bovine lens basement membrane.

Molecular transport through the basement membrane is important for a number of physiological functions, and dysregulation of basement membrane architecture can have serious pathological consequences. The structure-function relationships that govern molecular transport in basement membranes are not fully understood. The basement membrane from the lens capsule of the eye is a collagen IV-rich matrix that can easily be extracted and manipulated in vitro. As such, it provides a convenient model for studying the functional relationships that govern molecular transport in basement membranes. Here we investigate the effects of increased transmembrane pressure and solute electrical charge on the transport properties of the lens basement membrane (LBM) from the bovine eye. Pressure-permeability relationships in LBM transport were governed primarily by changes in diffusive and convective contributions to solute flux and not by pressure-dependent changes in intrinsic membrane properties. The solute electrical charge had a minimal but statistically significant effect on solute transport through the LBM that was opposite of the expected electrokinetic behavior. The observed transport characteristics of the LBM are discussed in the context of established membrane transport modeling and previous work on the effects of pressure and electrical charge in other basement membrane systems.

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.

Albumin handling by renal tubular epithelial cells in a microfluidic bioreactor.

Epithelial cells in the proximal tubule of the kidney reclaim and metabolize protein from the glomerular filtrate. Proteinuria, an overabundance of protein in the urine, affects tubular cell function and is a major factor in the progression of chronic kidney disease. By developing experimental systems to study tubular protein handling in a setting that simulates some of the environmental conditions of the kidney tubule in vivo, we can better understand how microenviromental conditions affect cellular protein handling to determine if these conditions are relevant in disease. To this end, we used two in vitro microfluidic models to evaluate albumin handling by renal proximal tubule cells. For the first system, cells were grown in a microfluidic channel and perfused with physiological levels of shear stress to evaluate the effect of mechanical stress on protein uptake. In the second system, a porous membrane was used to separate an apical and basolateral compartment to evaluate the fate of protein following cellular metabolism. Opossum kidney (OK) epithelial cells were exposed to fluorescently labeled albumin, and cellular uptake was determined by measuring the fluorescence of cell lysates. Confocal fluorescence microscopy was used to compare uptake in cells grown under flow and static conditions. Albumin processed by the cells was examined by size exclusion chromatography (SEC) and SDS-PAGE. Results showed that cellular uptake and/or degradation was significantly increased in cells exposed to flow compared to static conditions. This was confirmed by confocal microscopy. Size exclusion chromatography and SDS-PAGE showed that albumin was broken down into small molecular weight fragments and excreted by the cells. No trace of intact albumin was detectable by either SEC or SDS-PAGE. These results indicate that fluid shear stress is an important factor mediating cellular protein handling, and the microfluidic bioreactor provides a novel tool to investigate this process.

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.

Basal lamina secreted by MDCK cells has size- and charge-selective properties.

The role electrical charge plays in determining glomerular permeability to macromolecules remains unclear. If the glomerular basement membrane (GBM) has any significant role in permselectivity, physical principles would suggest a negatively charged GBM would reject similarly charged more than neutral species. However, recent in vivo studies with negative and neutral glomerular probes showed the opposite. Whether this observation is due to unique characteristics of the probes used or is a general physiological phenomenon remains to be seen. The goal of this study was to use the basement membrane deposited by Madin-Darby canine kidney epithelial cells as a simple model of a biologically derived, negatively charged filter to evaluate size- and charge-based sieving properties. Fluorescein isothiocyanate-labeled carboxymethylated Ficoll 400 (FITC-CM Ficoll 400) and amino-4-methyl-coumarin-labeled Ficoll 400 (AMC Ficoll 400) were used as negatively charged and neutral tracer molecules, respectively, during pressure-driven filtration. Streaming potential measurement indicated the presence of fixed, negative charge in the basal lamina. The sieving coefficient for neutral Ficoll 400 decreased by ∼0.0013 for each 1-Šincrement in solute radius, compared with a decrease of 0.0023 per Šfor the anionic Ficoll 400. In this system, molecular charge played a significant role in determining the sieving characteristics of the membrane, pointing to solute charge as a potential contributor to GBM permselectivity.

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.

Molecular conformation and filtration properties of anionic Ficoll.

The physiology of glomerular permselectivity remains mechanistically obscure, despite its importance in human disease. Although electrical contributions to glomerular permselectivity have long been considered important, two recent reports demonstrated enhanced glomerular permeability to anionic versus neutral polysaccharides. The interpretation of these observations is complicated by confounding of the effects of chemical modification on charge with effects on size and shape. In this report, neutral and anionic Ficoll are characterized by size-exclusion chromatography with online light scattering and viscometry and filtration through a highly defined anionic filtration membrane. Neutral and carboxymethylated Ficoll are nearly identical in size and conformation, yet carboxymethylated Ficoll is retained by an anionic membrane in excess of neutral Ficoll. This suggests that comparisons between clearances of neutral and carboxymethylated Ficoll may be a sensitive probe of electrostatic interactions independent of size and conformation.