Reassessment of Exosome Composition.

The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.

An arteriovenous mock circulatory loop and accompanying bond graph model for in vitro study of peripheral intravascular bioartificial organs.

BACKGROUND: Silicon nanopore membrane-based implantable bioartificial organs are dependent on arteriovenous implantation of a mechanically robust and biocompatible hemofilter. The hemofilter acts as a low-resistance, high-flow network, with blood flow physiology similar to arteriovenous shunts commonly created for hemodialysis access. A mock circulatory loop (MCL) that mimics shunt physiology is an essential tool for refinement and durability testing of arteriovenous implantable bioartificial organs and silicon blood-interfacing membranes. We sought to develop a compact and cost-effective MCL to replicate flow conditions through an arteriovenous shunt and used data from the MCL and swine to inform a bond graph mathematical model of the physical setup. METHODS: Flow physiology through bioartificial organ prototypes was obtained in the MCL and during extracorporeal attachment to swine for biologic comparison. The MCL was tested for stability overtime by measuring pressurewave variability over a 48-h period. Data obtained in vitro and extracorporeally informed creation of a bond graph model of the MCL. RESULTS: The arteriovenous MCL was a cost-effective, portable system that reproduced flow rates and pressures consistent with a pulsatile arteriovenous shunt as measured in swine. MCL performance was stable over prolonged use, providing a cost-effective simulator for enhanced testing of peripherally implanted bioartificial organ prototypes. The corresponding bond graph model recapitulates MCL and animal physiology, offering a tool for further refinement of the MCL system.

Wearable and implantable artificial kidney devices for end-stage kidney disease treatment: Current status and review.

BACKGROUND: Chronic kidney disease (CKD) is a major cause of early death worldwide. By 2030, 14.5 million people will have end-stage kidney disease (ESKD, or CKD stage 5), yet only 5.4 million will receive kidney replacement therapy (KRT) due to economic, social, and political factors. Even for those who are offered KRT by various means of dialysis, the life expectancy remains far too low. OBSERVATION: Researchers from different fields of artificial organs collaborate to overcome the challenges of creating products such as Wearable and/or Implantable Artificial Kidneys capable of providing long-term effective physiologic kidney functions such as removal of uremic toxins, electrolyte homeostasis, and fluid regulation. A focus should be to develop easily accessible, safe, and inexpensive KRT options that enable a good quality of life and will also be available for patients in less-developed regions of the world. CONCLUSIONS: Hence, it is required to discuss some of the limits and burdens of transplantation and different techniques of dialysis, including those performed at home. Furthermore, hurdles must be considered and overcome to develop wearable and implantable artificial kidney devices that can help to improve the quality of life and life expectancy of patients with CKD.

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 distributed solute model: an extended two-pore model with application to the glomerular sieving of Ficoll.

One of the many unresolved questions regarding the permeability of the glomerular filtration barrier is the reason behind the marked difference in permeability between albumin and polysaccharide probe molecules such as Ficoll and dextran of the same molecular size. Although the differences in permeability have been mainly attributed to charge effects, we have previously shown that this would require a highly charged filtration barrier, having a charge density that is ~10 times more than that on the albumin molecule. In this article, the classic two-pore model was extended by introducing size distributions on the solute molecules, making them conformationally flexible. Experimental sieving data for Ficoll from the rat glomerulus and from precision-made silicon nanopore membranes were analyzed using the model. For the rat glomerulus a small-pore radius of 36.2 Å and a geometric standard deviation (gSD) for the Ficoll size-distribution of 1.16 were obtained. For the nanopore membranes, a gSD of 1.24 and a small-pore radius of 43 Å were found. Interestingly, a variation of only ~16% in the size of the polysaccharide molecule is sufficient to explain the difference in permeability between albumin and Ficoll. Also, in line with previous data, the effects of applying a size distribution on the solute molecule are only evident when the molecular size is close to the pore size. Surely there is at least some variation in the pore radii, and, likely, the gSD obtained in the current study is an overestimation of the "true" variation in the size of the Ficoll molecule.

Innovations in Wearable and Implantable Artificial Kidneys.

More than 2 million people worldwide receive treatment for end-stage renal disease (ESRD). Current modalities of renal replacement therapy include in-center hemodialysis, peritoneal dialysis, home hemodialysis, and kidney transplantation. Patient survival has gradually increased during the past 2 decades and efforts continue to improve mortality and quality of life for patients with ESRD. Developments in sorbent technology, nanotechnology, and cell culture techniques provide promise for new innovations in ESRD management. New modalities currently in testing include wearable (WAKs) and implantable artificial kidneys (IAKs). The automated WAK (AWAK) and WAK are devices that have undergone small trials in humans. Additional study is needed before regulatory approval, coverage decisions, and widespread clinical implementation. The IAK is a biohybrid combining artificial filters and living cells currently in preclinical testing. These portable devices reduce the need for large quantities of water and continuous electrical supply. This could lower some barriers to home dialysis, making self-care renal replacement therapy more accessible and desirable. If widely successful, these devices could reduce the need to build and staff dialysis facilities, thus lowering health care costs associated with dialysis. The potential advantages and shortcomings of the AWAK, WAK, and IAK are described here.

Acute Kidney Injury as a Risk Factor for Delirium and Coma during Critical Illness.

RATIONALE: Acute kidney injury may contribute to distant organ dysfunction. Few studies have examined kidney injury as a risk factor for delirium and coma. OBJECTIVES: To examine whether acute kidney injury is associated with delirium and coma in critically ill adults. METHODS: In a prospective cohort study of intensive care unit patients with respiratory failure and/or shock, we examined the association between acute kidney injury and daily mental status using multinomial transition models adjusting for demographics, nonrenal organ failure, sepsis, prior mental status, and sedative exposure. Acute kidney injury was characterized daily using the difference between baseline and peak serum creatinine and staged according to Kidney Disease Improving Global Outcomes criteria. Mental status (normal vs. delirium vs. coma) was assessed daily with the Confusion Assessment Method for the ICU and Richmond Agitation-Sedation Scale. MEASUREMENTS AND MAIN RESULTS: Among 466 patients, stage 2 acute kidney injury was a risk factor for delirium (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.07-2.26) and coma (OR, 2.04; 95% CI, 1.25-3.34) as was stage 3 injury (OR for delirium, 2.56; 95% CI, 1.57-4.16) (OR for coma, 3.34; 95% CI, 1.85-6.03). Daily peak serum creatinine (adjusted for baseline) values were also associated with delirium (OR, 1.35; 95% CI, 1.18-1.55) and coma (OR, 1.44; 95% CI, 1.20-1.74). Renal replacement therapy modified the association between stage 3 acute kidney injury and daily peak serum creatinine and both delirium and coma. CONCLUSIONS: Acute kidney injury is a risk factor for delirium and coma during critical illness.

Silicon nanoporous membranes as a rigorous platform for validation of biomolecular transport models.

Microelectromechanical systems (MEMS), a technology that resulted from significant innovation in semiconductor fabrication, have recently been applied to the development of silicon nanopore membranes (SNM). In contrast to membranes fabricated from polymeric materials, SNM exhibit slit-shaped pores, monodisperse pore size, constant surface porosity, zero pore overlap, and sub-micron thickness. This development in membrane fabrication is applied herein for the validation of the XDLVO (extended Derjaguin, Landau, Verwey, and Overbeek) theory of membrane transport within the context of hemofiltration. In this work, the XDLVO model has been derived for the unique slit pore structure of SNM. Beta-2-microglobulin (B2M), a clinically relevant "middle molecular weight" solute in kidney disease, is highlighted in this study as the solute of interest. In order to determine interaction parameters within the XDLVO model for B2M and SNM, goniometric measurements were conducted, yielding a Hamaker constant of 4.61× 10-21 J and an acid-base Gibbs free energy at contact of 41 mJ/m2. The XDLVO model was combined with existing models for membrane sieving, with predictions of the refined model in good agreement with experimental data. Furthermore, the results show a significant difference between the XDLVO model and the simpler steric predictions typically applied in membrane transport. The refined model can be used as a tool to tailor membrane chemistry and maximize sieving or rejection of different biomolecules.

Clinical Use of the Urine Biomarker [TIMP-2] × [IGFBP7] for Acute Kidney Injury Risk Assessment.

Acute kidney injury (AKI) is a serious complication, commonly occurring in the critically ill population, with devastating short- and long-term consequences. Despite standardization of the definition and staging of AKI, early recognition remains challenging given that serum creatinine level is a marker, albeit imperfect, of kidney function and not kidney injury. Furthermore, the delay in increase in serum creatinine level after loss of glomerular filtration also prevents timely detection of decreased kidney function in patients with AKI. During the past decade, numerous clinical investigations have evaluated the utility of several biomarkers in the early diagnosis and risk stratification of AKI. In 2014, the US Food and Drug Administration approved the marketing of a test based on the combination of urine concentrations of tissue inhibitor of metalloproteinase 2 and insulin-like growth factor binding protein 7 ([TIMP-2] × [IGFBP7]) to determine whether certain critically ill patients are at risk for developing moderate to severe AKI. The optimal role of this biomarker in the diagnosis, management, and prognosis of AKI in different clinical settings requires further clarification. In this perspective, we summarize the biological actions of these 2 cell-cycle arrest biomarkers and present important considerations regarding the clinical application, interpretation, and limitations of this novel test for the early detection of AKI.

Optimal design of perturbations for individual two-compartment pharmacokinetic analysis.

We consider the optimal design of pharmacokinetic studies in patients that receive intermittent hemodialysis and intravenous antibiotic. Hemodialysis perturbs the pharmacokinetic system, providing additional opportunity for study. Designs that allocate measurements to occur exclusively during hemodialysis are shown to be viable alternatives to conventional designs, where all measurements occur outside of hemodialysis. Furthermore, hybrid designs with both conventional and intradialytic measurements have nearly double the efficiency of conventional designs. Convex optimal design and Monte Carlo techniques were used to simultaneously optimize hemodialysis event characteristics and sampling times, accounting for population pharmacokinetic heterogeneity. We also present several related methodological innovations.

Bridging Translation by Improving Preclinical Study Design in AKI.

Despite extensive research, no therapeutic interventions have been shown to prevent AKI, accelerate recovery of AKI, or reduce progression of AKI to CKD in patients. This failure in translation has led investigators to speculate that the animal models being used do not predict therapeutic responses in humans. Although this issue continues to be debated, an important concern that has not been addressed is whether improvements in preclinical study design can be identified that might also increase the likelihood of translating basic AKI research into clinical practice using the current models. In this review, we have taken an evidence-based approach to identify common weaknesses in study design and reporting in preclinical AKI research that may contribute to the poor translatability of the findings. We focused on use of N-acetylcysteine or sodium bicarbonate for the prevention of contrast-induced AKI and use of erythropoietin for the prevention of AKI, two therapeutic approaches that have been extensively studied in clinical trials. On the basis of our findings, we identified five areas for improvement in preclinical study design and reporting. These suggested and preliminary guidelines may help improve the quality of preclinical research for AKI drug development.

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.

The bioartificial kidney: current status and future promise.

The rapid understanding of the cellular and molecular bases of organ function and disease processes will be translated in the next decade into new therapeutic approaches to a wide range of clinical disorders, including acute and chronic renal failure. Central to these new therapies are the developing technologies of cell therapy and tissue engineering, which are based on the ability to expand stem or progenitor cells in tissue culture to perform differentiated tasks and to introduce these cells into the patient either via extracorporeal circuits or as implantable constructs. Cell therapy devices are currently being developed to replace the filtrative, metabolic, and endocrinologic functions of the kidney lost in both acute and chronic renal failure. This review summarizes the current state of development of a wearable or implantable bioartificial kidney. These devices have the promise to be combined to produce a wearable or implantable bioartificial kidney for full renal replacement therapy that may significantly diminish morbidity and mortality in patients with acute or chronic kidney disease.

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.

Achieving more frequent and longer dialysis for the majority: wearable dialysis and implantable artificial kidney devices.

The long-term survival for many chronic kidney failure patients who remain treated by dialysis in economically advanced countries remains similar to that of those with solid-organ malignancy, despite a disproportionate amount of health-care expenditure. As such, the current paradigm of three times weekly in-center hemodialysis for 4 h or shorter sessions needs to change to improve patient outcomes. Although more frequent and longer dialysis sessions have been reported to improve cardiovascular risk surrogates and short-term outcomes, these options are only practically available to a very small fraction of the total dialysis population. As such, radically new approaches are required to improve patient outcomes and quality of life for the majority of dialysis patients. Currently, two different approaches are being developed, wearable devices based on current dialysis techniques and more futuristic implantable devices modeled on the natural nephron.

Inhibition of Transforming Growth Factor-ß Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-ß (TGF-ß) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-ß receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-ß receptors possibly responsible for the improved differentiation, evaluate whether TGF-ß inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-ß signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-ß inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-ß signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-ß by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-ß signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.